Novel canola protein isolated

ABSTRACT

A novel canola protein isolate consisting predominantly of 2S canola protein and having equal to better solubility properties and improved clarity properties, has an increased proportion of 2S canola protein and a decreased proportion of 7S canola protein. The novel canola protein isolate is formed by heat treatment or isoelectric precipitation of aqueous supernatant from canola protein micelle formation and precipitation, to effect precipitation of 7S protein which is sedimented and removed. Alternatively, the novel canola protein isolate may be derived from a selective membrane procedure in which an aqueous canola protein solution containing 12S, 7S and 2S canola proteins is subjected to a first selective membrane technique to retain 12S and 7S canola proteins in a retentate, which is dried to provide a canola protein isolate consisting predominantly of 7S canola protein, and to permit 2S canola protein to pass through the membrane. The permeate is subjected to a second selective membrane technique to retain 2S canola protein and to permit low molecular weight contaminants to pass through the membrane, and the retentate from the latter membrane technique is dried.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 12/213,500 filed Jun. 20, 2008, which itself is acontinuation-in-part of copending U.S. patent application Ser. No.11/038,086 filed Jan. 21, 2005 which claims priority under 35 USC 119(e)from U.S. Provisional Patent Application No. 60/537,031 filed Jan. 20,2004.

FIELD OF INVENTION

The present invention relates to the production of a novel canolaprotein isolate and its use in aqueous solutions, including soft drinksand sports drinks.

BACKGROUND TO THE INVENTION

Canola oil seed protein isolates having protein contents of at least 100wt % (N×6.25) can be formed from oil seed meal by a process as describedin copending U.S. patent application Ser. No. 10/137,391 filed May 3,2002 (WO 02/089597), assigned to the assignee hereof and the disclosuresof which are incorporated herein by reference. The procedure involves amultiple step process comprising extracting canola oil seed meal usingan aqueous salt solution, separating the resulting aqueous proteinsolution from residual oil seed meal, increasing the proteinconcentration of the aqueous solution to at least about 200 g/L whilemaintaining the ionic strength substantially constant by using aselective membrane technique, diluting the resulting concentratedprotein solution into chilled water to cause the formation of proteinmicelles, settling the protein micelles to form an amorphous, sticky,gelatinous, gluten-like protein micellar mass (PMM), and recovering theprotein micellar mass from supernatant having a protein content of atleast about 100 wt % (N×6.25). As used herein, protein content isdetermined on a dry weight basis. The recovered PMM may be dried.

In one embodiment of the process, the supernatant from the PMM settlingstep is processed to recover canola protein isolate from thesupernatant. This procedure may be effected by initially concentratingthe supernatant using an ultrafiltration membrane and drying theconcentrate. The resulting canola protein isolate has a protein contentof at least about 90 wt %, preferably at least about 100 wt % (N×6.25).

The procedures described in U.S. patent application Ser. No. 10/137,391are essentially batch procedures. In copending U.S. patent applicationSer. No. 10/298,678 filed Nov. 19, 2002 (WO 03/043439), assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, there is described a continuous process for making canolaprotein isolates. In accordance therewith, canola oil seed meal iscontinuously mixed with an aqueous salt solution, the mixture isconveyed through a pipe while extracting protein from the canola oilseed meal to form an aqueous protein solution, the aqueous proteinsolution is continuously conveyed through a selective membrane operationto increase the protein content of the aqueous protein solution to atleast about 50 g/L, while maintaining the ionic strength substantiallyconstant, the resulting concentrated protein solution is continuouslymixed with chilled water to cause the formation of protein micelles, andthe protein micelles are continuously permitted to settle while thesupernatant is continuously overflowed until the desired amount of PMMhas accumulated in the settling vessel. The PMM is recovered from thesettling vessel and may be dried. The PMM has a protein content of atleast about 90 wt % (N×6.25), preferably at least about 100 wt %. Theoverflowed supernatant may be processed to recover canola proteinisolate therefrom, as described above.

Canola seed is known to contain about 10 to about 30 wt % proteins andseveral different protein components have been identified. Theseproteins include a 12S globulin, known as cruciferin, a 7S protein and a2S storage protein, known as napin. As described in copending U.S.patent application Ser. No. 10/413,371 filed Apr. 15, 2003 (WO03/088760), assigned to the assignee hereof and the disclosures of whichare incorporated herein by reference, the procedures described above,involving dilution of concentrated aqueous protein solution to form PMMand processing of supernatant to recover additional protein, lead to therecovery of isolates of different protein profiles.

In this regard, the PMM-derived canola protein isolate has a proteincomponent composition of about 60 to about 98 wt % of 7S protein, about1 to about 15 wt % of 12S protein and 0 to about 25 wt % of 2S protein.The supernatant-derived canola protein isolate has a protein componentcomposition of about 60 to about 95 wt % of 2S protein, about 5 to about40 wt % of 7S protein and 0 to about 5 wt % of 12S protein. Thus, thePMM-derived canola protein isolate is predominantly 7S protein and thesupernatant-derived canola protein isolate is predominantly 2S protein.As described in the aforementioned U.S. patent application Ser. No.10/413,371, the 2S protein has a molecular mass of about 14,000 daltons,the 7S protein has a molecular mass of about 145,000 daltons and the 12Sprotein has a molecular mass of about 290,000 daltons.

There has previously been described in Krzyzaniak et al in Nahrung (42)1998, Nr. 3/4, p. 201-204, the purification to homogenity of the 2Sstorage protein napin from rape seed and characterization of thesecondary structure and conformation stability of the protein. The napinwas isolated by extraction from rape seeds with buffer A (50 mM NaH₂PO₄,pH 7.0, 1 mM EDTA), followed by precipitation using (NH₄)₂SO₄,dissolution of the resulting pellet in buffer A, dialysis against thesame buffer and desalting by gel chromatography using a Sephadex G-50column. Fractions containing napin extract were collected andre-precipitated with (NH₄)₂SO₄. The resulting crude napin was dissolvedin buffer B (buffer A at pH 7.4), dialyzed against it, then loaded on toa CM-Sephadex C-50 column and eluted with a gradient of 0.15 to 0.35 MNaCl in buffer B. Fractions containing napin were pooled, precipitatedwith (NH₄)₂SO₄, redissolved in buffer A and dialyzed. Napin was furtherpurified by cation exchange HPLC using gradients up to 1 M NaCl at pH5.0. The fractions with λ_(max)=280 nm were collected, concentrated andanalyzed.

The applicants are aware of additional literature references describingthe laboratory preparation of samples of 2S canola protein, purified tohomogeneity, as follows:

-   Berot, S., Compoint, J. P., Larre, C., Malabat, C. and    Gueguen, J. 2005. Large scale purification of rapeseed proteins    (Brassica napus L). J. Chromatography B, 818: 35-42.-   Bhatty, R. S., McKenzie, S. L. and Finlayson, A. J. 1968. The    proteins of rapeseed (Brassica napus L.) soluble in salt solutions.    Can. J. Biochem., 46:1191-1197.-   Gehrig, P. M., Krzyzaniak, A., Barciszewski, J. and    Biemann, K. 1996. Mass spectrometric amino acid sequencing of a    mixture of seed storage proteins (napin) from Brassica napus,    products of a multigene family. Proc. Natl. Acad. Sci. U.S.A., 93:    3647-3652.-   Monsalve, R. I. and Rodriguez, R. 1990. Purification and    characterization of proteins from the 2S fraction from seeds of the    Brassicaceae family. J. Exper. Bot., 41(222): 89-94.-   Muren, E., Ek, B., Bjork, I. and Rask, L. 1996. Structural    comparison of the precursor and the mature form of napin, the 2S    storage protein in Brassica napus. Eur. J. Biochem., 242: 214-219.

The novel canola protein isolates provided herein are distinguished fromsuch disclosures in that the 2S predominated canola protein isolatesprovided herein always comprise a small quantity of 7S protein.

Canola is also known as rapeseed or oil seed rape.

SUMMARY OF INVENTION

It has now surprisingly been found that a novel canola protein isolate,having an increased proportion of 2S protein, preferably containing atleast about 85 wt % of 2S protein and having a reduced proportion of 7Sprotein, exhibits superior properties in aqueous solution to thesupernatant-derived canola protein isolate prepared following theprocedure of the aforementioned U.S. patent application Ser. No.10/137,391. In addition to equal or greater solubility at a variety ofpH values, the novel canola protein isolate provided herein is able toprovide improved clarity in solution with soft drinks and sports drinks,providing clear protein fortified beverages.

In addition, the novel canola protein isolate exhibits a greatersolubility than the supernatant-derived canola protein isolate at a pHof about 3.5 in about 0.1M sodium chloride solution.

In general, the present invention, in its broadest aspect, provides acomposition comprising a first isolated or purified protein component ofa plant origin and a second isolated or purified protein component of aplant origin; wherein, upon heat treatment of an about 1% w/v dispersionof the composition in water at a pH of between about 6 to about 7 atabout 85° C. for about 10 minutes, less than about 15% of thecomposition is insoluble.

In a preferred aspect of the present invention, the composition, uponsuch heat treatment, has less than about 10% of the composition isinsoluble, more preferably substantially none of the composition isinsoluble.

Preferably, the plant is canola, the first protein component comprises a2S canola protein and the second protein component comprises a 7S canolaprotein. The 2S canola component preferably comprises at least about 85wt % of the composition, more preferably at least 95 wt % of thecomposition. In a preferred embodiment, the 7S canola protein comprisesat least 2 wt % of the composition.

The composition of the invention generally has a protein content of atleast about 90 wt % (N×6.25), preferably at least about 100 wt %.

The first protein component and the second protein component may nothave been hydrolyzed.

The composition of the present invention is particularly useful as abeverage component to provide a protein fortified beverage. Accordingly,in another aspect of the invention, there is provided a beveragecomprising the composition of the invention.

The beverage composition of the invention preferably has a pH in therange of about 2.5 to about 5, which is the range for most commonbeverages. Thus, the composition of the invention may be used to provideprotein fortification for common beverages. In some cases, modificationto the normal formulation of the beverage to tolerate the composition ofthe invention may be necessary where components present in the beveragemay adversely affect the ability of the composition of the invention toremain dissolved in the beverage. The composition of the presentinvention also may be used for protein fortification of neutral pHbeverage.

In a preferred aspect of the present invention, 12 fluid ounces of thebeverage contain at least 5 gram of the composition.

In another aspect of the present invention, there is provided an aqueoussolution of the composition of the invention. Such aqueous solutionpreferably has a clarity of an about 1 wt % aqueous solution over a pHrange of about 2 to about 7 of less than about 1 when determined bymeasuring absorbance of visible light at 600 nm.

In another aspect of the present invention, there is provided a beveragecomprising an isolated or purified protein of vegetable origin, wherein,upon heat treatment of an about 1% w/v dispersion of the protein inwater at a pH of about 6 to about 7 at about 85° C. for 10 minutes, lessthan about 15 wt % of the protein is insoluble. Preferably, upon suchheat treatment, substantially none of the protein is insoluble. In apreferred aspect of the invention, the isolated or purified proteinprovides at least about 5 grams of protein per serving, which may be 12ounces of beverage.

In an additional aspect of the present invention, there is provided abeverage comprising an isolated and purified protein of vegetableorigin, wherein an about 1% w/v aqueous solution of the protein has aclarity on a pH range of about 2 to about 7 of less than about 1 whendetermined by measuring absorbance of visible light at 600 nm. Theisolated or purified protein comprises at least about 5 grams of proteinper serving.

The protein of vegetable origin preferably is canola.

In another aspect of the present invention, there is provided acomposition having a protein content of at least about 90 wt % (N×6.25)on a dry weight basis (d.b.) and comprising a first canola proteinisolate component which is the 2S protein of canola, and a second canolaprotein isolate component which is the 7S protein of canola, thecomposition being formed by processing the aqueous supernatant fromcanola protein micelle formation and precipitation and having asolubility in aqueous media greater than a canola protein isolatederived directly from the aqueous supernatant from the canola proteinmicelle formation and precipitation, wherein the solubility isdetermined as described in Example 15 and/or Example 18.

Such composition preferably has a number of preferred properties, asfollows:

-   -   a surface hydrophobicity of less than about 25, as determined in        Example 19, below    -   an amino acid profile substantially as set forth in the column        of Table XXVI below headed C200H    -   an absorbance at 280 nm, determined as described in Example 20        below, which is less than the canola protein derived directly        from the aqueous supernatant.    -   upon heat treatment of an about 1% w/v dispersion of the        composition in water at a pH of about 6 to about 7 at about        85° C. for 10 minutes, less than 15% of the composition is        insoluble, preferably substantially none of the composition is        insoluble.

The 2S protein of canola preferably comprises at least about 85 wt % ofthe composition, more preferably at least about 95 wt % of thecomposition. The 7S protein of canola preferably comprises at leastabout 2 wt % of the composition.

In an additional aspect of the invention, there is provided an aqueoussolution of the composition having a protein content of at least about90 wt % (N×6.25) on a dry weight basis (d.b.) and comprising a firstcanola protein isolate component which is the 2S protein of canola, anda second canola protein isolate component which is the 7S protein ofcanola, the composition being formed by processing the aqueoussupernatant from canola protein micelle formation and precipitation andhaving a solubility in aqueous media greater than a canola proteinisolate derived directly from the aqueous supernatant from the canolaprotein micelle formation and precipitation, wherein the solubility isdetermined as described in Example 15 and/or Example 18.

The aqueous solution preferably has clarity of an about 1 wt % aqueoussolution over a pH range of about 2 to about 7 of less than about 1 whendetermined by measuring absorbance of visible light at 600 nm.

In addition, the present invention provides a beverage comprising acomposition having a protein content of at least about 90 wt % (N×6.25)on a dry weight basis (d.b.) and comprising a first canola proteinisolate component which is the 2S protein of canola and a second canolaprotein isolate component which is the 7S protein of canola, thecomposition being formed by processing the aqueous supernatant fromcanola protein micelle formation and precipitation and having asolubility in aqueous media greater than a canola protein isolatederived directly from the aqueous supernatant from the canola proteinmicelle formation and precipitation, wherein the solubility isdetermined as described in Example 15 and/or Example 18.

The beverage preferably has a pH of about 2.5 to about 5. 12 fluidounces of such beverage preferably comprises at least about 5 grams ofthe composition.

In a further aspect of the present invention, there is provided a canolaprotein isolate consisting predominantly of 2S canola protein having aprotein content of at least about 90 wt % (N×6.25) on a dry weight basis(d.b.) and having an increased proportion of 2S canola protein and adecreased proportion of 7S canola protein when compared to canolaprotein isolates consisting predominantly of 2S canola protein andderived from aqueous supernatant from canola protein micelle formationand precipitation.

In an additional aspect of the present invention, there is provided acanola protein isolate having a protein content of at least about 90 wt% (N×6.25) on a dry weight basis (d.b.) and containing at least about 85wt % of 2S canola protein and less than about 15 wt % of 7S canolaprotein of the canola proteins present in the isolate.

The novel canola protein isolate and composition of the presentinvention possess a number of unique properties, different from those ofthe supernatant derived canola protein isolate and include solubility,clarity, heat stability, absorbance at 280 nm, surface hydrophobicityand amino acid profile. These properties are described below and thedata supporting this discussion appear in the Examples below.

Solubility:

The novel canola protein isolate exhibits the same or increasedsolubility in water and in carbonated and non-carbonated soft drinks andsport drinks in comparison to supernatant-derived canola protein isolateover a wide range of pH and at about 1% w/v protein concentration.

In addition, the novel canola protein isolates exhibits the same orincreased solubility in water over a wide range of pH and at an about 1%w/v protein concentration, wherein, following formation of the solutionsor dispersions, the samples are centrifuged to sediment insolublematerial and yield a clear supernatant.

Further, the novel canola protein isolate exhibits greater solubility in0.1M sodium chloride solution at pH 3.5 in comparison tosupernatant-derived canola protein isolate.

Accordingly, an aspect of the present invention provides a canolaprotein isolate having a protein content of at least about 90 wt %(N×6.25) on a dry material basis (d.b.) and containing at least about 85wt % of 2S canola protein, and the canola protein isolate having asolubility in aqueous media greater than a canola protein isolatederived from the aqueous supernatant from canola protein micelleformulation and precipitation when the solubility is determined asdescribed in Example 15 and/or Example 18 below.

Clarity:

The novel canola protein isolate may be incorporated into a variety ofbeverages, including carbonated and non-carbonated soft drinks as wellas juices, punches and cocktails, to provide protein fortification tosuch beverages. Such beverages have a wide range of pH values, rangingfrom about 2.5 to about 5, and often are packaged in 12 fluid ouncequantities. The novel canola protein isolate may be added in anyconvenient quantity to provide protein fortification to the beverage,for example, at least about 5 g of the novel canola protein isolate per12 fluid ounce quantity. The canola protein isolate may be blended withdried beverage prior to reconstitution of the beverage by dissolution inwater.

The added novel canola protein isolate dissolves in the beverage anddoes not impair the clarity of the beverage, even after thermalprocessing, such as in hot fill applications. In a clear beverage at aprotein concentration of 2 wt %, the clarity is less than about 0.5 whendetermined by measuring the absorbance of visible light at 600 nm. Inwater at a protein concentration of 1 wt %, the clarity is less thanabout 1.0 over a pH range of about 2 to about 7, when determined bymeasuring the absorbance of visible light at 600 nm.

Accordingly, in another aspect of the present invention, there isprovided an aqueous composition comprising a canola protein isolatehaving a protein content of at least about 90 wt % (N×6.25) on a dryweight basis (d.b.) and containing at least about 85 wt % of 2S canolaprotein dissolved in an aqueous medium, the aqueous medium being:

-   -   (a) water and having a clarity of a 1 wt % aqueous solution of        the novel canola protein isolate over a pH range of about 2 to        about 7 of less than about 1 when determined by measuring        absorbance of visible light at 600 nm, or    -   (b) an aqueous beverage and having a clarity of a 2 wt % aqueous        solution of the novel canola protein isolate of less than about        0.5 when determined by measuring the absorbance of visible light        at 600 nm.

Heat Stability:

The novel canola protein isolate of the invention has improved heatstability both at pH 6 and 7 in comparison to supernatant-derived canolaprotein isolate. Following heat treatment of the supernatant toprecipitate 7S protein therefrom, as described in detail below, theheat-treated supernatant is resistant to further protein deposition.Accordingly, another aspect of the present invention provides a canolaprotein isolate having a protein content of at least about 90 wt %(N×6.25) d.b. and containing at least about 85 wt % of 2S protein whichcanola protein isolate is heat stable to precipitation of protein whenheated as a 1 wt % aqueous solution thereof at a pH of 6 or 7 for 10minutes at 85° C.

Absorbance at 280 nm:

The novel canola protein of the invention has weaker absorbance at 280nm than supernatant-derived canola protein isolate. This resultindicates that the novel canola protein isolate of the inventioncontains less tyrosine and tryptophan, which absorb strongly at 280 nm,than supernatant-derived canola protein.

Accordingly, in another aspect of the present invention, there isprovided a canola protein isolate having a protein content of at leastabout 90 wt % (N×6.25) on a dry weight basis (d.b.) and containing atleast about 85 wt % of 2S canola protein having an absorbance at 280 nm,determined as described in Example 20 below, which is less than a canolaprotein isolate derived from the aqueous supernatant from canola proteinmicelle formation and precipitation.

Surface Hydrophobicity:

The novel canola protein isolate of the invention has a lower surfacehydrophobicity than supernatant-derived canola protein isolate, probablyarising from increased globulin content of the supernatant-derivedcanola protein isolate than the novel protein isolate.

The surface hydrophobicity of the novel canola protein isolate isgenerally less than about 25, preferably less than about 20.

Accordingly, in another aspect of the invention, there is provided acanola protein isolate having a protein content of at least about 90 wt% (N×6.25) on a dry weight basis having a surface hydrophobicity,determined as described in Example 19 below, of less than about 25,preferably less than about 20.

Amino Acid Profile:

The novel canola protein isolate of the invention exhibits an amino acidprofile which is different from that of supernatant-derived canolaprotein isolate, exhibiting increased levels of cysteine, proline,lysine and glutamine-glutamate and decreased levels of tyrosine andaspargine-aspartate.

Accordingly, in another aspect of the present invention, there isprovided a canola protein isolate having a protein content of at leastabout 90 wt % (N×6.25) on a dry weight basis having an amino acidprofile substantially as set forth in the column of Table XXVI headedC200H.

The novel canola protein isolate of the invention may be prepared bythermal treatment of the concentrated supernatant from the procedure ofU.S. patent application Ser. No. 10/137,391 in order to reduce theproportion of 7S protein in the concentrated supernatant and hence toincrease the proportion of 2S protein. Alternatively, the heat treatmentmay be carried out on the supernatant prior to concentration orfollowing partial concentration of the supernatant. Accordingly, inanother aspect of the present invention, there is provided a process forthe preparation of a canola protein isolate having an increasedproportion of 2S canola protein, which comprises (a) providing anaqueous solution of 2S and 7S proteins consisting predominantly of 2Sprotein, (b) heat treating the aqueous solution to cause precipitationof 7S canola protein, (c) removing precipitated 7S protein from theaqueous solution, and (d) recovering a canola protein isolate having aprotein content of at least about 90 wt % (N×6.25) d.b. and having anincreased proportion of 2S canola protein.

Alternatively, the novel canola protein isolate may be prepared by aprocedure in which, following extraction of protein from the canola oilseed meal, the protein solution is subjected to a first selectivemembrane step with a membrane having a molecular weight cut-off whichpermits the 2S protein to pass through the membrane in a permeate whilethe 7S and 12S proteins are retained in a retentate. The retentate thenis dried to provide a first canola protein isolate which ispredominantly 7S protein. The permeate from the first selective membraneprocess step is then subjected to a second selective membrane step witha membrane having a molecular weight cut-off which retains the 2Sprotein and permits low molecular weight contaminants, including salt,phenolics and anti-nutritional materials, to pass through. The retentatefrom the latter selective membrane step then is dried to provide asecond canola protein isolate which is predominantly 2S protein andwhich is the novel protein isolate.

Accordingly, in an additional aspect of the present invention, there isprovided a process for the preparation of a canola protein isolate,which comprises (a) providing an aqueous canola protein solution derivedfrom canola oil seed meal and containing 12S, 7S and 2S canola proteins,(b) increasing the protein concentration of the aqueous solution using aselective membrane technique which is effective to retain 7S and 12Scanola proteins in a retentate and to permit 2S protein to pass throughthe membrane as a permeate to provide a concentrated protein solution,(c) drying the retentate from step (b) to provide a canola proteinisolate consisting predominantly of 7S canola protein and having aprotein content of at least about 90 wt % (N×6.25) on a dry weight basis(d.b.), (d) increasing the concentration of the permeate from step (b)using a selective membrane technique which is effective to retain 2Scanola protein in a retentate and to permit low molecular weightcontaminants to pass through the membrane in a permeate, and (e) dryingthe retentate from step (d) to provide a canola protein isolateconsisting predominantly of 2S protein and having a protein content ofat least about 90 wt % (N×6.25) d.b.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a schematic representation of a protein solution recoveryprocess according to embodiments of the invention; and

FIG. 2 is a schematic representation of the procedure which may be usedto produce the novel canola protein isolate of the present inventionfrom the supernatant from the PMM-forming process shown in FIG. 1.

GENERAL DESCRIPTION OF INVENTION

The novel canola protein isolate provided herein has a protein contentof at least about 90 wt % (N×6.25), preferably at least about 100 wt %,and may be isolated from canola oil seed meal by a batch process, or acontinuous process, or a semi-continuous process.

The novel canola protein isolate provided herein consists predominantlyof 2S protein and has an increased proportion of 2S canola protein and adecreased proportion of 7S canola protein when compared to canolaprotein isolates consisting predominantly of 2S protein and derived fromsupernatant from canola protein micelle formation and precipitation andprepared under the same experimental conditions of preparation. As setforth above, the novel canola protein isolates of the invention, whilehaving a decreased proportion of 7S protein and an increased proportionof 2S protein, there is always present a small residual quantity of 7Sprotein.

The novel canola protein isolates contain at least about 85 wt % of 2Scanola protein and less than about 15 wt % of 7S canola protein,preferably at least about 90 wt % of 2S canola protein and less thanabout 10 wt % of 7S canola protein and more preferably as great aproportion of 2S protein as is possible. As noted above, such canolaprotein isolate may be obtained by heat treatment of supernatant,partially concentrated supernatant and concentrated supernatant, asdescribed in more detail below. The heat treatment of the supernatant,partially concentrated supernatant and concentrated supernatant causesprecipitation of the 7S protein, which can be removed from theheat-treated supernatant by any convenient means, such as centrifugationor filtration. The 2S protein is not affected by the heat treatment andhence the heat treatment increases the proportion of 2S protein presentby decreasing the proportion of 7S protein.

The novel canola protein isolate is soluble in aqueous solution over awide range of pH values, generally about pH 2 to about pH 7.5,preferably about 2 to about 4, generally having solubility equal to orgreater than canola protein isolate consisting predominantly of 2Sprotein and derived from supernatant from canola protein micelleformation and precipitation under the same experimental conditions ofpreparation. In addition, aqueous solutions of the novel canola proteinisolate in soft drinks, including both carbonated and non-carbonatedsoft drinks and sports drinks, including both carbonated andnon-carbonated sports energy drinks, such as thosecommercially-available, have a greater clarity than such aqueoussolutions produced from canola protein isolate consisting predominantlyof 2S protein and derived from supernatant from canola protein micelleformation and precipitation under the same conditions of preparation.

The concentration of canola protein isolate in the aqueous solution,including solution in soft drinks and sports drinks, may vary dependingon the intended use of the solution. In general, the proteinconcentration may vary from about 0.1 to about 30 wt %, preferably about1 to about 5 wt %.

Accordingly, the present invention includes aqueous solutions of thenovel canola protein isolate provided herein, including not only thosementioned above, but also other beverages, such as juices, alcoholicbeverages, coffee-based beverages and dairy-based beverages.

The initial step of the process of providing canola protein isolatesinvolves solubilizing proteinaceous material from canola oil seed meal.The proteinaceous material recovered from canola seed meal may be theprotein naturally occurring in canola seed or the proteinaceous materialmay be a protein modified by genetic manipulation but possessingcharacteristic hydrophobic and polar properties of the natural protein.The canola meal may be any canola meal resulting from the removal ofcanola oil from canola oil seed with varying levels of non-denaturedprotein, resulting, for example, from hot hexane extraction or cold oilextrusion methods. The removal of canola oil from canola oil seedusually is effected as a separate operation from the protein isolaterecovery procedure described herein.

Protein solubilization is effected most efficiently by using a foodgrade salt solution since the presence of the salt enhances the removalof soluble protein from the oil seed meal. Where the canola proteinisolate is intended for non-food uses, non-food-grade chemicals may beused. The salt usually is sodium chloride, although other salts, suchas, potassium chloride, may be used. The salt solution has an ionicstrength of at least about 0.05, preferably at least about 0.10, toenable solubilization of significant quantities of protein to beeffected. As the ionic strength of the salt solution increases, thedegree of solubilization of protein in the oil seed meal initiallyincreases until a maximum value is achieved. Any subsequent increase inionic strength does not increase the total protein solubilized. Theionic strength of the food grade salt solution which causes maximumprotein solubilization varies depending on the salt concerned and theoil seed meal chosen.

In view of the greater degree of dilution required for proteinprecipitation with increasing ionic strengths, it is usually preferredto utilize an ionic strength value less than about 0.8, and morepreferably a value of about 0.1 to about 0.15.

In a batch process, the salt solubilization of the protein is effectedat a temperature of from about 5° C. to about 75° C., preferablyaccompanied by agitation to decrease the solubilization time, which isusually about 10 to about 60 minutes. It is preferred to effect thesolubilization to extract substantially as much protein from the oilseed meal as is practicable, so as to provide an overall high productyield.

The lower temperature limit of about 5° C. is chosen sincesolubilization is impractically slow below this temperature while theupper preferred temperature limit of about 75° C. is chosen due to thedenaturation temperature of some of the present proteins.

In a continuous process, the extraction of the protein from the canolaoil seed meal is carried out in any manner consistent with effecting acontinuous extraction of protein from the canola oil seed meal. In oneembodiment, the canola oil seed meal is continuously mixed with a foodgrade salt solution and the mixture is conveyed through a pipe orconduit having a length and at a flow rate for a residence timesufficient to effect the desired extraction in accordance with theparameters described herein. In such continuous procedure, the saltsolubilization step is effected rapidly, in a time of up to about 10minutes, preferably to effect solubilization to extract substantially asmuch protein from the canola oil seed meal as is practicable. Thesolubilization in the continuous procedure is effected at temperaturesbetween about 10° C. and about 75° C., preferably between about 15° C.and about 35° C.

The aqueous food grade salt solution generally has a pH of about 5 toabout 6.8, preferably about 5.3 to about 6.2, the pH of the saltsolution may be adjusted to any desired value within the range of about5 to about 6.8 for use in the extraction step by the use of anyconvenient acid, usually hydrochloric acid, or alkali, usually sodiumhydroxide, as required.

The concentration of oil seed meal in the food grade salt solutionduring the solubilization step may vary widely. Typical concentrationvalues are about 5 to about 15% w/v.

The protein extraction step with the aqueous salt solution has theadditional effect of solubilizing fats which may be present in thecanola meal, which then results in the fats being present in the aqueousphase.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 40 g/L, preferably about 10 toabout 30 g/L.

The aqueous salt solution may contain an antioxidant. The antioxidantmay be any convenient antioxidant, such as sodium sulfite or ascorbicacid. The quantity of antioxidant employed may vary from about 0.01 toabout 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit oxidation of phenolics in the proteinsolution.

The aqueous phase resulting from the extraction step then may beseparated from the residual canola meal, in any convenient manner, suchas by employing a decanter centrifuge, followed by disc centrifugationand/or filtration to remove residual meal. The separated residual mealmay be dried for disposal.

The colour of the final canola protein isolate can be improved in termsof light colour and less intense yellow by the mixing of powderedactivated carbon or other pigment adsorbing agent with the separatedaqueous protein solution and subsequently removing the adsorbent,conveniently by filtration, to provide a protein solution. Diafiltrationalso may be used for pigment removal.

Such pigment removal step may be carried out under any convenientconditions, generally at the ambient temperature of the separatedaqueous protein solution, employing any suitable pigment adsorbingagent. For powdered activated carbon, an amount of about 0.025% to about5% w/v, preferably about 0.05% to about 2% w/v, is employed.

Where the canola seed meal contains significant quantities of fat, asdescribed in U.S. Pat. Nos. 5,844,086 and 6,005,076, assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, then the defatting steps described therein may be effected onthe separated aqueous protein solution and on the concentrated aqueousprotein solution discussed below. When the colour improvement step iscarried out, such step may be effected after the first defatting step.

As an alternative to extracting the oil seed meal with an aqueous saltsolution, such extraction may be made using water alone, although theutilization of water alone tends to extract less protein from the oilseed meal than the aqueous salt solution. Where such alternative isemployed, then the salt, in the concentrations discussed above, may beadded to the protein solution after separation from the residual oilseed meal in order to maintain the protein in solution during theconcentration step described below. When a first fat removal step iscarried out, the salt generally is added after completion of suchoperations.

Another alternative procedure is to extract the oil seed meal with thefood grade salt solution at a relatively high pH value above about 6.8,generally up to about 9.9. The pH of the food grade salt solution may beadjusted to the desired alkaline value by the use of any convenientfood-grade alkali, such as aqueous sodium hydroxide solution.Alternatively, the oil seed meal may be extracted with the salt solutionat a relatively low pH below about pH 5, generally down to about pH 3.Where such alternative is employed, the aqueous phase resulting from theoil seed meal extraction step then is separated from the residual canolameal, in any convenient manner, such as by employing decantercentrifugation, followed by disc centrifugation and/or filtration toremove residual meal. The separated residual meal may be dried fordisposal.

The aqueous protein solution resulting from the high or low pHextraction step then is pH adjusted to the range of about 5 to about6.8, preferably about 5.3 to about 6.2, as discussed above, prior tofurther processing as discussed below. Such pH adjustment may beeffected using any convenient acid, such as hydrochloric acid, oralkali, such as sodium hydroxide, as appropriate.

The aqueous protein solution may be processed in two alternativeprocedures, depending on whether 7S-rich protein micellar mass is to beprecipitated to leave a supernatant for processing to form the novelcanola protein isolate, or the aqueous protein solution is to beprocessed by a two-membrane operation without precipitation of proteinmicellar mass to obtain the novel canola protein isolate.

In the first alternative procedure, the aqueous protein solution isconcentrated to increase the protein concentration thereof whilemaintaining the ionic strength thereof substantially constant. Suchconcentration generally is effected to provide a concentrated proteinsolution having a protein concentration of at least about 50 g/L,preferably at least about 200 g/L, more preferably at least about 250g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 100,000 daltons, preferably about 5,000 to about10,000 daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass through themembrane while preventing higher molecular weight species from so doing.The low molecular weight species include not only the ionic species ofthe food grade salt but also low molecular weight materials extractedfrom the source material, such as, carbohydrates, pigments andanti-nutritional factors, as well as any low molecular weight forms ofthe protein. The molecular weight cut-off of the membrane is usuallychosen to ensure retention of a significant proportion of the protein inthe solution, while permitting contaminants to pass through havingregard to the different membrane materials and configurations.

The concentrated protein solution then may be subjected to adiafiltration step using an aqueous salt solution of the same molarityand pH as the extraction solution. Such diafiltration may be effectedusing from about 2 to about 20 volumes of diafiltration solution,preferably about 5 to about 10 volumes of diafiltration solution. In thediafiltration operation, further quantities of contaminants are removedfrom the aqueous protein solution by passage through the membrane withthe permeate. The diafiltration operation may be effected until nosignificant further quantities of contaminants and visible colour arepresent in the permeate. Such diafiltration may be effected using thesame membrane as for the concentration step. However, if desired, thediafiltration step may be effected using a separate membrane with adifferent molecular weight cut-off, such as a membrane having amolecular weight cut-off in the range of about 3,000 to about 100,000daltons, preferably about 5,000 to about 10,000 daltons, having regardto different membrane materials and configuration.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit oxidationof phenolics present in the concentrated canola protein isolatesolution.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 20° to about 60° C., preferablyabout 20 to about 30° C., and for the period of time to effect thedesired degree of concentration. The temperature and other conditionsused to some degree depend upon the membrane equipment used to effectthe concentration and the desired protein concentration of the solution.

The concentrated and optionally diafiltered protein solution may besubject to a further defatting operation, if required, as described inU.S. Pat. Nos. 5,844,086 and 6,005,076.

The concentrated and optionally diafiltered protein solution may besubject to a colour removal operation as an alternative to the colourremoval operation described above. Powdered activated carbon may be usedherein as well as granulated activated carbon (GAC). Another materialwhich may be used as a colour adsorbing agent is polyvinyl pyrrolidone.

The colour adsorbing agent treatment step may be carried out under anyconvenient conditions, generally at the ambient temperature of thecanola protein solution. For powdered activated carbon, an amount ofabout 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v,may be used. Where polyvinylpyrrolidone is used as the colour adsorbingagent, an amount of about 0.5% to about 5% w/v, preferably about 2% toabout 3% w/v, may be used. The colour adsorbing agent may be removedfrom the canola protein solution by any convenient means, such as byfiltration.

The concentrated and optionally diafiltered protein solution resultingfrom the optional colour removal step may be subjected to reduce themicrobial load. Such pasteurization may be effected under any desiredpasteurization conditions. Generally, the concentrated and optionallydiafiltered protein solution is heated to a temperature of about 55° toabout 70° C., preferably about 60° to about 65° C., for about 10 toabout 15 minutes, preferably about 10 minutes. The pasteurizedconcentrated protein solution then may be cooled for further processingas described below, preferably to a temperature of about 25° to about40° C.

Depending on the temperature employed in the concentration step andoptional diafiltration step and whether or not a pasteurization step iseffected, the concentrated protein solution may be warmed to atemperature of at least about 20°, and up to about 60° C., preferablyabout 25° to about 40° C., to decrease the viscosity of the concentratedprotein solution to facilitate performance of the subsequent dilutionstep and micelle formation. The concentrated protein solution should notbe heated beyond a temperature above which micelle formation does notoccur on dilution by chilled water.

The concentrated protein solution resulting from the concentration step,and optional diafiltration step, optional colour removal step, optionalpasteurization step and optional defatting step, then is diluted toeffect micelle formation by mixing the concentrated protein solutionwith chilled water having the volume required to achieve the degree ofdilution desired. Depending on the proportion of canola protein desiredto be obtained by the micelle route and the proportion from thesupernatant, the degree of dilution of the concentrated protein solutionmay be varied. With lower dilution levels, in general, a greaterproportion of the canola protein remains in the aqueous phase.

When it is desired to provide the greatest proportion of the protein bythe micelle route, the concentrated protein solution is diluted by about5 fold to about 25 fold, preferably by about 10 fold to about 20 fold.

The chilled water with which the concentrated protein solution is mixedhas a temperature of less than about 15° C., generally about 1° to about15° C., preferably less than about 10° C., since improved yields ofprotein isolate in the form of protein micellar mass are attained withthese colder temperatures at the dilution factors used.

In a batch operation, the batch of concentrated protein solution isadded to a static body of chilled water having the desired volume, asdiscussed above. The dilution of the concentrated protein solution andconsequential decrease in ionic strength causes the formation of acloud-like mass of highly associated protein molecules in the form ofdiscrete protein droplets in micellar form. In the batch procedure, theprotein micelles are allowed to settle in the body of chilled water toform an aggregated, coalesced, dense, amorphous sticky gluten-likeprotein micellar mass (PMM). The settling may be assisted, such as bycentrifugation. Such induced settling decreases the liquid content ofthe protein micellar mass, thereby decreasing the moisture contentgenerally from about 70% by weight to about 95% by weight to a value ofgenerally about 50% by weight to about 80% by weight of the totalmicellar mass. Decreasing the moisture content of the micellar mass inthis way also decreases the occluded salt content of the micellar mass,and hence the salt content of dried isolate.

Alternatively, the dilution operation may be carried out continuously bycontinuously passing the concentrated protein solution to one inlet of aT-shaped pipe, while the diluting water is fed to the other inlet of theT-shaped pipe, permitting mixing in the pipe. The diluting water is fedinto the T-shaped pipe at a rate sufficient to achieve the desireddegree of dilution of the concentrated protein solution.

The mixing of the concentrated protein solution and the diluting waterin the pipe initiates the formation of protein micelles and the mixtureis continuously fed from the outlet from the T-shaped pipe into asettling vessel, from which, when full, supernatant is permitted tooverflow. The mixture preferably is fed into the body of liquid in thesettling vessel in a manner which minimizes turbulence within the bodyof liquid.

In the continuous procedure, the protein micelles are allowed to settlein the settling vessel to form an aggregated, coalesced, dense,amorphous, sticky, gluten-like protein micellar mass (PMM) and theprocedure is continued until a desired quantity of the PMM hasaccumulated in the bottom of the settling vessel, whereupon theaccumulated PMM is removed from the settling vessel. In lieu of settlingby sedimentation, the PMM may be separated continuously bycentrifugation.

The combination of process parameters of concentrating of the proteinsolution to a preferred protein content of at least about 200 g/L andthe use of a dilution factor of about 10 to about 20, result in higheryields, often significantly higher yields, in terms of recovery ofprotein in the form of protein micellar mass from the original mealextract, and much purer isolates in terms of protein content thanachieved using any of the known prior art protein isolate formingprocedures discussed in the aforementioned US patents.

By the utilization of a continuous process for the recovery of canolaprotein isolate as compared to the batch process, the initial proteinextraction step can be significantly reduced in time for the same levelof protein extraction and significantly higher temperatures can beemployed in the extraction step. In addition, in a continuous operation,there is less chance of contamination than in a batch procedure, leadingto higher product quality and the process can be carried out in morecompact equipment.

The settled isolate is separated from the residual aqueous phase orsupernatant, such as by decantation of the residual aqueous phase fromthe settled mass or by centrifugation. The PMM may be used in the wetform or may be dried, by any convenient technique, such as spray dryingor freeze drying, to a dry form. The dry PMM has a high protein content,in excess of about 90 wt % protein, preferably at least about 100 wt %protein (calculated as N×6.25), and is substantially undenatured (asdetermined by differential scanning calorimetry). The dry PMM isolatedfrom fatty oil seed meal also has a low residual fat content, when theprocedures of U.S. Pat. Nos. 5,844,086 and 6,005,076 are employed asnecessary, which may be below about 1 wt %.

As described in the aforementioned U.S. patent application Ser. No.10/413,371, the PMM consists predominantly of a 7S canola protein havinga protein component composition of about 60 to 98 wt % of 7S protein,about 1 to about 15 wt % of 12S protein and 0 to about 25 wt % of 2Sprotein.

The supernatant from the PMM formation and settling step containssignificant amounts of canola protein, not precipitated in the dilutionstep, and is processed to recover canola protein isolate therefrom. Asdescribed in the aforementioned U.S. patent application Ser. No.10/413,371, the canola protein isolate derived from the supernatantconsists predominantly of 2S canola protein having a protein componentcontent of about 60 to about 95 wt % of 2S protein, about 5 to about 40wt % of a 7S protein and 0 to about 5 wt % of 12S protein.

The supernatant from the dilution step, following removal of the PMM, isconcentrated to increase the protein concentration thereof. Suchconcentration is effected using any convenient selective membranetechnique, such as ultrafiltration, using membranes with a suitablemolecular weight cut-off permitting low molecular weight species,including the salt and other non-proteinaceous low molecular weightmaterials extracted from the protein source material, to pass throughthe membrane, while retaining canola protein in the solution.Ultrafiltration membranes having a molecular weight cut-off of about3,000 to 100,000 daltons, preferably about 5,000 to about 10,000daltons, having regard to differing membrane materials andconfiguration, may be used. Concentration of the supernatant in this wayalso reduces the volume of liquid required to be dried to recover theprotein. The supernatant generally is concentrated to a proteinconcentration of at least about 50 g/L, preferably about 100 to about300 g/L, more preferably about 200 to about 300 g/L, prior to drying.Such concentration operation may be carried out in a batch mode or in acontinuous operation, as described above for the protein solutionconcentration step.

The concentrated supernatant then may be subjected to a diafiltrationstep using water, saline or acidified water. Such diafiltration may beeffected using from about 2 to about 20 volumes of diafiltrationsolution, preferably about 5 to about 10 volumes of diafiltrationsolution. In the diafiltration operation, further quantities ofcontaminants are removed from the aqueous supernatant by passage throughthe membrane with the permeate. The diafiltration operation may beeffected until no significant further quantities of contaminants andvisible colour are present in the permeate. Such diafiltration may beeffected using the same membrane as for the concentration step. However,if desired, the diafiltration may be effected using a separate membrane,such as a membrane having a molecular weight cut-off in the range ofabout 3,000 to about 100,000 daltons, preferably about 5,000 to about10,000 daltons, having regard to different membrane materials andconfiguration.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit oxidationof phenolics present in the concentrated canola protein isolatesolution.

The concentrated and optionally diafiltered protein solution may besubject to a colour removal operation as an alternative to the colourremoval operation described above. Powdered activated carbon may be usedherein as well as granulated activated carbon (GAC). Another materialwhich may be used as a colour adsorbing agent is polyvinyl pyrrolidone.

The colour adsorbing agent treatment step may be carried out under anyconvenient conditions, generally at the ambient temperature of thecanola protein solution. For powdered activated carbon, an amount ofabout 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v,may be used. Where polyvinylpyrrolidone is used as the colour adsorbingagent, an amount of about 0.5% to about 5% w/v, preferably about 2% toabout 3% w/v, may be used. The colour adsorbing agent may be removedfrom the canola protein solution by any convenient means, such as byfiltration.

In accordance with the present invention, the concentrated andoptionally diafiltered supernatant, following the optional colourremoval operation, is heat treated to decrease the quantity of the 7Sprotein present in the solution by precipitation and removal of the 7Sprotein and thereby increasing the proportion of 2S protein in thecanola protein present in the concentrated supernatant.

Such heat treatment may be effected using a temperature and time profilesufficient to decrease the proportion of 7S present in the concentratedsupernatant, preferably to reduce the proportion of 7S protein by asignificant extent. In general, the 7S protein content of thesupernatant is reduced by at least about 50 wt %, preferably at leastabout 75 wt % by the heat treatment. In general, the heat treatment maybe effected at a temperature of about 70° to about 120° C., preferablyabout 75° to about 105° C., for about 1 second to about 30 minutes,preferably about 5 to about 15 minutes. The precipitated 7S protein maybe removed in any convenient manner, such as centrifugation orfiltration or a combination thereof.

The concentrated heat-treated supernatant may be acidified prior todrying, to a pH corresponding to the intended use of the dried isolate,generally a pH down to about 2 to about 5, preferably about 2.5 to about4.

The concentrated heat-treated supernatant, after removal of theprecipitated 7S protein, such as by centrifugation, may be dried by anyconvenient technique, such as spray drying or freeze drying, to a dryform to provide a canola protein isolate in accordance with the presentinvention. Such novel canola protein isolate has a high protein content,in excess of about 90 wt %, preferably at least about 100 wt % protein(calculated as N×6.25) and is expected to be substantially undenatured.

Such novel canola protein isolate contains a high proportion of 2Sprotein, preferably at least 90 wt % and most preferably at least about95 wt %, of the canola protein in the isolate. There is also aproportion of 7S protein in the isolate

Alternatively, the heat treatment of the supernatant to precipitate 7Sprotein may be effected on the supernatant prior to the concentrationand diafiltration steps mentioned above. Following removal of thedeposited 7S protein, the supernatant then is concentrated, optionallydiafiltered, optionally submitted to a colour removal operation, anddried to provide the canola protein isolate according to the invention.

As a further alternative, the supernatant first may be partiallyconcentrated to any convenient level. The partially concentratedsupernatant then is subjected to the heat treatment to precipitate 7Sprotein. Following removal of the precipitated 7S protein, thesupernatant is further concentrated, generally to a concentration ofabout 50 to about 300 g/L, preferably about 200 to about 300 g/L,optionally diafiltered, optionally submitted to a colour removaloperation, and dried to provide the canola protein isolate according tothe invention.

Precipitated 7S protein is removed from the supernatant, partiallyconcentrated supernatant or concentrated supernatant by any convenientmeans, such as centrifugation or filtration or a combination thereof.

Following removal of precipitated 7S protein, the heat treatedsupernatant or partially concentrated, heat treated supernatant may beacidified at any point during or after concentration or diafiltration,as discussed above.

In another embodiment of the invention, the supernatant from the micelleformation and precipitation is processed in an alternative manner toform the novel canola protein isolate of the invention. The supernatantmay further be first concentrated or partially concentrated, asdiscussed above.

A salt, usually sodium chloride, although other salts such as potassiumchloride may be used, first is added to the supernatant, partiallyconcentrated supernatant or concentrated supernatant to provide asalinated solution having a conductivity of at least about 0.3 mS,preferably about 10 to about 20 mS.

The pH of the salinated supernatant is adjusted to a value to causeisoelectric precipitation of 7S protein, generally to a pH of about 2.0to about 4.0, preferably about 3.0 to about 3.5. The isoelectricprecipitation of the 7S protein may be effected over a wide temperaturerange, generally from about 5° C. to about 70° C., preferably about 10°C. to about 40° C. The precipitated 7S protein is removed from theisoelectrically precipitated supernatant by any convenient means, suchas centrifugation or filtration or a combination thereof.

The isoelectrically precipitated supernatant, if not alreadyconcentrated, then is concentrated as discussed above and diafiltered toremove the salt, prior to drying the concentrated and diafilteredsupernatant to form the canola protein isolate of the invention. Theconcentrated and diafiltered supernatant may be filtered to removeresidual particulates and subjected to an optional colour removal step,as discussed above, prior to drying by any convenient technique, such asspray drying or freeze drying, to a dry form to provide a canola proteinisolate according to the present invention. Such canola protein isolatehas a high protein content, in excess of about 90 wt %, preferably atleast about 100 wt % protein (N×6.25).

In the second alternative procedure to produce the novel canola proteinisolate, the aqueous protein solution produced by extraction of thecanola oil seed protein meal is concentrated to increase the proteinconcentration thereof while maintaining the ionic strength thereofsubstantially constant by a first ultrafiltration step using membranes,such as hollow-fibre membranes or spiral wound membranes, having amolecular weight cut-off sufficient to retain the 7S and 12S proteins ina retentate and to permit 2S protein to pass through the membrane. Asuitable molecular weight cut-off range for the membrane is from about30,000 to about 1,000,000 daltons, preferably about 50,000 to about100,000 daltons having regard to differing membrane materials andconfigurations. For continuous operation, the membranes are dimensionedto permit the desired degree of concentration as the aqueous proteinsolution passes through the membranes.

The first ultrafiltration step may be effected to concentrate theaqueous protein solution from about 4 to about 20 fold to a proteinconcentration of at least about 50 g/L, preferably at least about 200g/L and more preferably at least about 250 g/L.

The concentrated protein solution preferably then is subjected to adiafiltration step using an aqueous salt solution of the same molarityand pH as the extraction solution. An antioxidant may be present in thediafiltration medium during at least part of the diafiltration step toinhibit oxidation of phenolics in the concentrated canola proteinisolate solution. The antioxidant may be any convenient antioxidant,such as sodium sulfite or ascorbic acid. The quantity of antioxidantemployed in the diafiltration medium depends on the material employedand may vary from about 0.01 to about 1 wt %, preferably about 0.05 wt%.

The diafiltration step may be effected by using from about 2 to about 20volumes of diafiltration solution, preferably about 5 to about 10volumes of diafiltration solution. During the diafiltration operation,2S protein, phenolics and visible colour components along with other lowmolecular weight components are removed from the concentrated proteinsolution by passage through the membrane with the permeate.

The diafiltration step may be effected using the same membrane as usedfor the concentration step.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 20° to about 60° C., preferablybelow about 30° C., and for a period of time to effect the desireddegree of concentration and washing. The temperatures and otherconditions used depend to some degree on the membrane equipment used toeffect the concentration and the desired protein concentration of thesolution.

The membrane used in the first ultrafiltration step permits asignificant proportion of the 2S protein to pass into the permeate,along with other low molecular weight species, including the ionicspecies of the food grade salt, carbohydrates, phenolics, pigments andanti-nutritional factors. The molecular weight cut-off is normallychosen to ensure retention of a significant proportion of the 7S and 12Sprotein in the retentate, while permitting the 2S protein andcontaminants to pass through, having regard to the different membranematerials and configurations.

The retentate from the concentration step and optional diafiltrationstep then is dried by any convenient technique, such as spray drying orfreeze drying, to a dry form. The dried protein has a high proteincontent, in excess of about 90 wt % protein, preferably at least about100 wt % protein (N×6.25), and is expected to be substantiallyundenatured. The dried protein isolate consists predominantly of thecanola 7S protein, with some 12S protein and possibly small quantitiesof 2S protein. In general, the dried canola protein isolate contains:

about 60 to about 95 wt % of 7S protein

about 2 to about 15 wt % of the 12S protein

0 to about 30 wt % of the 2S protein.

Preferably, the dried canola protein isolate contains:

about 70 to about 95 wt % of 7S protein

about 5 to about 10 wt % of the 12S protein

0 to about 20 wt % of the 2S protein.

The permeate from the concentration step and optional diafiltration stepis concentrated in a second ultrafiltration step using membranes, suchas hollow-fibre membranes or spiral wound membranes, having a suitablemolecular weight cut-off to retain the 2S protein while permitting lowmolecular weight species, including salt, phenolics, colour componentsand anti-nutritional factors, to pass through the membrane.Ultrafiltration membranes having a molecular weight cut-off of about3,000 to about 30,000 daltons, preferably about 5,000 to about 10,000daltons, having regard to differing membrane materials andconfigurations, may be used. The permeate generally is concentrated to aprotein concentration of at least about 50 g/L, preferably about 100 toabout 300 g/L, more preferably about 200 to about 300 g/L, prior todrying. Such a concentration operation may be carried out in a batchmode or in a continuous operation, as described above for the proteinsolution concentration step.

The concentrated permeate may be subjected to a diafiltration step usingwater. An antioxidant may be present in the diafiltration medium duringat least part of the diafiltration step to inhibit oxidation ofphenolics in the concentrated permeate. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe material employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %.

The diafiltration step may be effected using 2 to 20 volumes ofdiafiltration solution, preferably about 5 to about 10 volumes ofdiafiltration solution. In the diafiltration operation furtherquantities of contaminants and visible colour components are removedfrom the concentrated permeate by passage through the diafiltrationmembrane. The diafiltration operation may be effected until nosignificant further quantities of contaminants and visible colourcomponents are removed in the permeate.

The diafiltration step may be effected using the same membrane as usedin the concentration step. Alternatively, a separate membrane may beused having a molecular weight cut-off in the range of about 3,000 toabout 30,000 daltons, preferably about 5,000 to about 10,000 daltons,having regard to different membrane materials and configurations.

The concentrated and optionally diafiltered permeate may be acidified atany point during or after concentration and prior to drying, asdiscussed above.

The concentrated and optionally diafiltered permeate is dried by anyconvenient technique, such as spray drying or freeze drying, to a dryform. The dried protein has a high protein content, in excess of about90 wt % protein, preferably at least about 100 wt % (N×6.25), and isexpected to be substantially undenatured.

If desired, a portion of the concentrated canola protein isolate fromthe first ultrafiltration step may be combined with a portion of theconcentrated permeate from the second ultrafiltration step prior todrying the combined streams by any convenient technique to provide acombined canola protein isolate composition. The relative proportions ofthe proteinaceous materials mixed together may be chosen to provide aresulting canola protein isolate composition having a desired profile of2S/7S/12S proteins. Alternatively, the dried protein isolates may becombined in any desired proportion to provide any desired specific2S/7S/12S protein profile in the mixtures. The combined canola proteinisolate composition has a high protein content, in excess of about 90 wt%, preferably at least about 100 wt % (calculated as N×6.25), and isexpected to be substantially undenatured.

By operating in this manner, a number of canola protein isolates may berecovered as dry mixtures of various proportions by weight of firstultrafiltration-derived canola protein isolate and secondultrafiltration-derived canola protein isolate, generally about 5:95 toabout 95:5 by weight, which may be desirable for attaining differingfunctional and nutritional properties based on the differing proportionsof 2S/7S/12S proteins in the compositions.

The dried canola protein isolates provided herein following theprocedures described above consist predominantly of the canola 2Sprotein with small quantities of 7S protein. In general, the driedcanola protein isolate contains:

about 85 to less than 100 wt % of 2S protein,

preferably about 90 to less than 100 wt % of 2S protein,

up to about 15 wt % of 7S protein,

preferably up to about 10 wt % of 7S protein.

DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to FIG. 1, there is shown therein the novel two-membraneprocess provided in accordance with one aspect of the invention incomparison to the formation of canola protein isolates (CPIs) by themicelle route.

As can be seen, retentate from a first ultrafiltration stage(Ultrafiltration #1), which may comprise ultrafiltration anddiafiltration steps, is processed in one of two ways. In thetwo-membrane embodiment of the invention, the retentate is spray driedto provide a canola protein isolate which consists predominantly of 7Scanola protein.

In the procedure of the aforementioned U.S. patent application Ser. No.10/137,321, the retentate is passed to a dilution step in which canolaprotein isolate is precipitated as a protein micellar mass. The proteinmicellar mass is spray dried to provide a canola protein isolateconsisting predominantly of 7S canola protein.

In the two-membrane embodiment of the invention, the permeate from thefirst ultrafiltration step is subjected to a second ultrafiltration step(Ultrafiltration #2-A), which may include ultrafiltration anddiafiltration. The retentate from the second ultrafiltration step isspray dried to provide a canola protein isolate consisting predominantlyof 2S protein.

In the procedure of U.S. Ser. No. 10/137,321, the supernatant from theprecipitation of the protein micellar mass is subjected to anultrafiltration step (Ultrafiltration #2-B), which may includeultrafiltration and diafiltration. The retentate from theultrafiltration step is spray dried to provide a canola protein isolateconsisting predominantly of the 2S protein.

Referring to FIG. 2, there is shown therein a procedure for processingthe supernatant from the micelle process to produce the novel canolaprotein isolate of the invention wherein heat treatment or isoelectricprecipitation of the supernatant is effected, either followingconcentration (Ultrafiltration #2) (right-hand flow) or prior toconcentration (left-hand flow).

EXAMPLES Example 1

This Example describes the production of a novel canola protein isolatein accordance with one embodiment of the invention.

An ‘a’ kg of canola meal was added to ‘b’ L of 0.1 M NaCl solution atambient temperature and agitated for 30 minutes to provide an aqueousprotein solution. The residual canola meal was removed and the resultingprotein solution was clarified by centrifugation and filtration toproduce ‘c’ L of filtered protein solution having a protein content of‘d’ % by weight.

A ‘e’ L aliquot of the protein extract solution was reduced in volume to‘f’ L by concentration on a polyvinylidene difluoride (PVDF) membranehaving a molecular weight cutoff of 5,000 daltons and then diafilteredwith ‘g’ L of 0.1M NaCl solution on the same membrane. The diafilteredretentate was then pasteurized at 60° C. for 10 minutes. The resultingpasteurized concentrated protein solution had a protein content of ‘h’ %by weight.

The concentrated solution at ‘i’° C. was diluted ‘j’ into cold RO waterhaving a temperature ‘k’ ° C. A white cloud formed immediately and wasallowed to settle. The upper diluting water was removed and theprecipitated, viscous, sticky mass (PMM) was recovered from the bottomof the vessel in a yield of ‘l’ wt % of the filtered protein solution.The dried PMM derived protein was found to have a protein content of ‘m’% (N×6.25) d.b. The product was given a designation ‘n’ C300.

The parameters ‘a’ to ‘n’ for two runs are set forth in the followingTable I:

TABLE I n BW-SA034-J12-04A BW-SA035-J14-04A a 15 15 b 150 150 c 75 68 d1.93 1.95 e 75 68 f 4 4 g 20 20 h 19.08 14.20 i 33 33 j 1:10 1:10 k 3 3l 37.45 28.32 m 103.08 99.73

The removed supernatant was reduced in volume to ‘o’ L byultrafiltration using a polyethersulfone (PES) membrane having amolecular weight cut-off of 10,000 daltons and then the concentrate wasdiafiltered on the same membrane with ‘p’ L of water. The diafilteredconcentrate was then pasteurized at 60° C. for 10 minutes. Thepasteurized concentrate contained ‘q’ % protein by weight. With theadditional protein recovered from the supernatant, the overall proteinrecovery of the filtered protein solution was ‘r’ wt %. The pasteurizedconcentrate was split into two equal portions. One portion was spraydried to form a final product given designation ‘n’ C200 and had aprotein content of ‘s’ % (N×6.25) d.b.

The parameters ‘n’ to ‘s’ for two runs are set forth in the followingTable II:

TABLE II n BW-SA034-J12-04A BW-SA035-J14-04A o 3.5 3 p 7 6 q 4.83 4.30 r49.35 38.05 s 91.73 93.69

The other portion of the pasteurized, concentrated supernatant washeated to 85° C. for 10 minutes and then centrifuged to removeprecipitated protein. The resulting concentrate was then spray dried toform a final product given designation ‘n’ C200H and had a proteincontent of ‘t’ % (N×6.25). The parameters ‘n’ and ‘t’ for two runs areset forth in the following Table III:

TABLE III n BW-SA034-J12-04A BW-SA035-J14-04A t 91.32 92.11

Example 2

This Example shows the effect of heating temperature and time on theprotein profile of canola protein isolate produced from concentratedsupernatant.

A solution of C200 canola protein isolate from batch SA035-J14-04A,prepared as described in Example 1, was prepared in reverse osmosispurified water to a protein concentration of 5 wt %. The solution wasprepared by stirring the protein and water with a magnetic stir bar forone hour at room temperature.

Samples of the protein solution (25 ml) were heated in centrifuge tubesin a temperature controlled water bath. Samples were heated for 10minutes at a temperature of 75, 80, 85, 90 or 95° C. Timing started whenthe internal temperature of the sample was within 1° C. of the desiredlevel and the samples were mixed constantly throughout the heatingprocess. After heat treatment, the samples were centrifuged at 8,000 gfor 10 minutes and the supernatants analyzed for protein profile basedon peak area by size exclusion HPLC.

The results obtained are set forth in the following Table IV:

TABLE IV Protein profiles of C200 solutions heated to differenttemperatures Treatment temperature % 7S % 2S Control (no heat) 22.6 77.475° C. 5.3 94.7 80° C. 3.6 96.4 85° C. 3.5 96.5 90° C. 1.8 98.2 95° C.1.8 98.2

As may be seen from the results set forth in Table IV, all the heattreatments applied resulted in a significant reduction in 7S proteinfrom the samples. Treatment at 90° C. resulted in the lowest level of 7Sand no additional improvement was gained by raising the temperature to95° C. It was determined by size exclusion HPLC analysis that the heattreatment had little effect on the peak area for 2S. This means that theheat treatment resulted in minimal loss of 2S protein.

A sample of protein solution (80 ml) was heated in a jacketed vesselattached to a circulating water bath set to a temperature of 90° C. andthe sample was mixed with a magnetic stir bar. Aliquots of heatedsolution were removed after 5, 10 and 15 minutes of heating time. Timingdid not start until the temperature of the sample measured 85° C. Afterheat treatment, the collected samples were centrifuged at 8,000 g for 10minutes and the supernatants analyzed for protein profile based on eacharea by size exclusion HPLC.

The results obtained are set forth in the following Table V:

TABLE V Protein profiles of C200 solutions heated for different lengthsof time Treatment time % 7S % 2S Control (no heat) 22.6 77.4  5 min 1.898.2 10 min 1.9 98.1 15 min 1.9 98.1

As may be seen from the results set forth in Table V, there was nosignificant difference in the level of 7S protein in the samples for anyof the tested times.

As is apparent from the results outlined in this Example, a significantreduction in the level of 7S protein in concentrated supernatant can beobtained with a wide variety of heating conditions.

Example 3

This Example contains an evaluation of protein profiles for the canolaprotein isolates produced according to Example 1.

Size exclusion HPLC was used to evaluate the protein profile based onpeak area of the concentrated supernatant and modified concentratedsupernatant produced according to the procedures of Example 1. The spraydried products were dissolved at a 1 wt % level in 0.1 M NaCl prior toHPLC analysis.

The results obtained are set forth in the following Table VI:

TABLE VI % Batch Product 12S % 7S % 2S SA034-J12-04A Concentratedsupernatant (C200) 0.00 13.13 86.87 SA034-J12-04A Modified concentrated0.00 3.55 96.45 supernatant (C200H) SA035-J14-04A Concentratedsupernatant (C200) 0.00 24.52 75.48 SA035-J14-04A Modified concentrated0.00 7.55 92.45 supernatant (C200H)

As may be seen from the results set forth in Table VI, the heattreatment of the concentrated supernatant results in a significantreduction of the quantity of 7S protein in the spray dried canolaprotein isolate compared to the absence of such heat treatment.

Example 4

This Example describes the production of a novel canola protein isolatein accordance with embodiments of the invention.

Canola meal was added to sodium chloride solution at ambient temperatureand agitated for 30 minutes to provide an aqueous protein solution. Theresidual canola meal was removed and the resulting protein was clarifiedby centrifugation and filtration.

An aliquot of the protein extract solution was reduced in volume byconcentration on a polyvinylidene difluoride (PVDF) membrane and thenoptionally diafiltered with sodium chloride solution on the samemembrane. The retentate was then pasteurized at 60° C. for 10 minutes.

The concentrated solution was diluted into cold RO water. A white cloudformed immediately and was allowed to settle. The upper diluting waterwas removed and the precipitated, viscous, sticky mass (PMM) wasrecovered either by decantation or by centrifugation. The PMM was spraydried to form a final product.

Heat treatment was then carried out on the removed diluting water(termed the supernatant), partially concentrated supernatant orconcentrated supernatant.

In the case where the heat treatment was carried out on the concentratedsupernatant, ‘a’ L of the supernatant was reduced in volume to ‘b’ L byultrafiltration using a polyethersulfone (PES) membrane having amolecular weight cut-off of ‘c’ Daltons and then the concentrate wasdiafiltered on the same membrane with ‘d’ L of water. The diafilteredconcentrate was then pasteurized at 60° C. for 10 minutes. Thepasteurized concentrate contained ‘e’ % protein by weight. With theadditional protein recovered from the supernatant, the overall proteinrecovery of the filtered protein solution was ‘f’ wt %. The pasteurizedconcentrate was split into two portions. One portion of ‘g’ L was spraydried to form a final product given designation ‘h’ C200 and had aprotein content of ‘i’ % (N×6.25) d.b.

The parameters ‘a’ to ‘i’ for six runs are set forth in the followingTable VII:

TABLE VII BW-SA033- BW-MS034- BW-SD061- BW-SD062- BW-SD061- BW-SD062- hE10-04A E04-05A K07-05A L18-05A I26-06A K20-06A a 755 36 33 451 n/a n/ab 38 2.9 4.5 45 n/a n/a c 100,000 10,000 10,000 10,000 n/a n/a d 0 723.5 225 n/a n/a e 15.2 5.7 6.18 8.73 n/a n/a f 53.7 51.8 66.7 58.4 n/an/a g 38 1.45 0 0 n/a n/a i 97.12 95.23 n/a n/a n/a n/a

The other portion of ‘j’ L of the pasteurized concentrated supernatantwas heated to 85° C. for 10 minutes and then centrifuged to removeprecipitated protein. The resulting centrate was then spray dried toform a final product given designation ‘h’ C200H and had a proteincontent of ‘k’ % (N×6.25) d.b. The parameters ‘h, ‘j’ and ‘k’ for sixruns are set forth in the following Table VIII:

TABLE VIII BW-SA033- BW-MS034- BW-SD061- BW-SD062- BW-SD061- BW-SD062- hE10-04A E04-05A K07-05A L18-05A I26-06A K20-06A j 0 1.45 4.5 45 n/a n/ak n/a 92.9 95.49 96.86 n/a n/a

In the case where the heat treatment was carried out on the supernatantor partially concentrated supernatant, ‘l’ L of supernatant was reducedin volume to ‘m’ L by ultrafiltration using a polyethersulfone (PES)membrane having a molecular weight cut-off of ‘n’ Daltons. Thesupernatant or partially concentrated supernatant was then heated to 85°C. for 10 minutes and then centrifuged to remove precipitated protein.When necessary, residual precipitated protein was removed by filtration.The clarified sample was then reduced in volume to ‘o’ L byultrafiltration using a polyethersulfone (PES) membrane having amolecular weight cut-off of ‘p’ Daltons. The concentrate contained ‘q’ %protein by weight. With the additional protein recovered from thesupernatant, the overall protein recovery of the filtered proteinsolution was ‘r’ wt %. The concentrate was spray dried to form a finalproduct given designation ‘h’ C200HS and had a protein content of ‘s’ %(N×6.25) d.b. The parameters ‘h’ and ‘l’ to ‘s’ for six runs are setforth in the following Table IX:

TABLE IX BW-SA033- BW-MS034- BW-SD061- BW-SD062- BW-SD061- BW-SD062- hE10-04A E04-05A K07-05A L18-05A I26-06A K20-06A l n/a n/a n/a n/a 670908 m n/a n/a n/a n/a 670 228 n n/a n/a n/a n/a n/a 10,000 o n/a n/a n/an/a 26.4 25.6 p n/a n/a n/a n/a 10,000 10,000 q n/a n/a n/a n/a 11.612.57 r n/a n/a n/a n/a 59.3 57.5 s n/a n/a n/a n/a 92.38 92.42

Example 5

This Example contains an evaluation of protein profiles for the canolaprotein isolates produced according to Example 4.

Size exclusion HPLC was used to evaluate the protein profile (based onpeak area) of the concentrated supernatant and modified productsproduced according to the procedures of Example 4. The spray driedproducts were dissolved at a 1 wt % level in 0.1 M NaCl prior to HPLCanalysis.

The results obtained are set forth in the following Table X:

TABLE X % Batch Product 12S % 7S % 2S SA033-E10-04A Concentratedsupernatant (C200) 0.00 32.18 67.82 MS043-E04-05A Concentratedsupernatant (C200) 1.34 20.19 78.46 Modified concentrated 1.6 6.25 92.15supernatant (C200H) SD061-K07-05A Modified concentrated 1.05 5.95 93.00supernatant (C200H) SD062-L18-05A Modified concentrated 1.24 9.24 89.52supernatant (C200H) SD061-I26-06A Supernatant heat treated 1.02 4.0594.93 then concentrated (C200HS) SD062-K20-06A Supernatant partially1.02 4.95 94.03 concentrated, then heat treated, then concentrated(C200HS)

As may be seen from the results set forth in Table X, the heat treatmentresulted in a significant reduction of the quantity of 7S protein in thespray dried canola protein isolate compared to the absence of such heattreatment.

Example 6

This Example describes the production of a novel canola protein isolatein accordance with one embodiment of the invention.

Canola meal was added to sodium chloride solution at ambient temperatureand agitated for 30 minutes to provide an aqueous protein solution. Theresidual canola meal was removed and the resulting protein solution wasclarified by centrifugation and filtration.

An aliquot of the protein extract solution was reduced in volume byconcentration on a polyethersulfone (PES) membrane. The retentate wasthen pasteurized at 60° C. for 1 minute.

The concentrated solution was diluted into cold RO water. A white cloudformed immediately. The precipitated, viscous, sticky mass (PMM) wasseparated from the supernatant by centrifugation. The PMM was spraydried to form a final product.

“a” L of supernatant was combined with “b” kg of sodium chloride toadjust the salt concentration to about 0.1 M. Following salt addition,the pH was adjusted to 3.5 by adding dilute hydrochloric acid. Thesample was allowed to rest for 10 minutes as protein precipitationoccurred.

The precipitated protein was removed by centrifugation from theacidified supernatant. The precipitated protein was spray dried to forma final product.

The pH of the acidified supernatant was lowered to 3 by the addition ofdilute hydrochloric acid. “c” L acidified supernatant was then reducedin volume to about “d” L by ultrafiltration using a polyethersulfone(PES) membrane having a molecular weight cut-off of “e” Daltons and thenthe concentrate was diafiltered on the same membrane with “f” L ofacidified water. The diafiltered concentrate was filtered to remove anyresidual precipitated protein. An aliquot of “g” kg was spray dried toform a final product given designation “h” C200IS, which had a proteincontent of “i” wt % (N×6.25) d.b.

The parameters “a” to “i” are set forth in the following Table XI:

TABLE XI h BW-SA077-K07-07A a 111.2 b 0.65 c 95 d 7 e 10,000 f 30 g 4 i92.86

Example 7

This Example contains an evaluation of the solubility of the canolaprotein isolates produced in Example 1.

The solubility of the spray dried concentrated supernatant (C200) andmodified concentrated supernatant (C200H) produced by the procedures ofExample 1, was determined using a modified version of the procedure ofMorr et al, J. Food Sci. 50:1715-1718.

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (4, 5,6 or 7) with NaOH or HCl. A sample was also prepared at native pH. Forthe pH adjusted samples, the pH was measured and corrected two timesduring the 60 minutes stirring. After the 60 minutes of stirring, thesample was made up to 50 ml total volume with RO water, yielding a 1%w/v protein dispersion. An aliquot of the protein dispersion wasreserved for protein content determination by Leco analysis using a LecoFA28 Nitrogen Determinator. Another portion of the sample wascentrifuged at 8000 g for 10 minutes. This sedimented any undissolvedmaterial and yielded a clear supernatant. The protein content of thesupernatant was then determined by Leco analysis.

Solubility (%)=(Supernatant protein conc./Original dispersion proteinconc.)×100

The results obtained are set forth in the following Table XII:

TABLE XII Solubility (%) Batch Product pH 4 pH 5 pH 6 pH 7 Native pHSA034-J12-04A Concentrated supernatant 88.1 100 86.1 97.3 89.8SA034-J12-04A Modified concentrated supernatant 100 100 100 100 100SA035-J14-04A Concentrated supernatant 87.4 95 90.6 90.6 86.7SA035-J14-04A Modified concentrated supernatant 100 100 100 100 100

As can be seen from the results of Table XII, the dried isolate frommodified concentrated supernatant (C200H) was notably more soluble inwater at various pH values than the dried isolate from concentratedsupernatant (C200).

Example 8

This Example contains an evaluation of the solubility of the canolaprotein isolates produced in Examples 4 and 6.

The solubility of the spray dried concentrated supernatant (C200) andmodified products (C200H and C200HS) produced by the procedures ofExample 4 and isoelectrically precipitated supernatant (C200IS) producedby the procedures of Example 6, was determined using a modified versionof the procedure of Morr et al., J. Food. Sci. 50:1715-1718, asdescribed above in Example 7, except with the pH range extended to 2 to7 along with native pH.

The results obtained are set forth in the following Table XIII:

TABLE XIII Solubility (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7Native pH SA033- Concentrated 86.5 86.7 91.7 97.2 81.2 75.6 89.8 E10-supernatant (C200) 04A MS043- Concentrated 87.4 81.5 92.4 95.3 93.8 94.289.8 E04- supernatant (C200) 05A MS043- Modified 100 100 100 91.7 92.590.7 94.3 E04- concentrated 05A supernatant (C200H) SD061- Modified 96.795.7 100 97.1 95.7 98.6 86.6 K07- concentrated 05A supernatant (C200H)SD062- Modified 98.8 89.0 97.9 100 94.1 100 100 L18- concentrated 05Asupernatant (C200H) SD061- Supernatant heat 97.7 92.4 100 95.9 100 98.494.3 I26- treated then 06A concentrated (C200HS) SD062- Supernatantpartially 100 94.1 100 91.8 100 100 90.6 K20- concentrated, then 06Aheat treated, then concentrated (C200HS) SA027- Isoelectrically 100 98.0100 95.7 100 100 100 K07- precipitated 07A supernatant (C200IS)

As can be seen from the results of Table XIII, the dried isolates frommodified concentrated supernatant (C200H), heat treated thenconcentrated supernatant (C200HS) and isoelectrically precipitatedsupernatant (C200IS), were notably more soluble in water at various pHvalues than the dried isolate from concentrated supernatant (C200).

Example 9

This Example contains an evaluation of the solubility of the canolaprotein isolates produced in Example 1 in a soft drink.

The solubility in a soft drink (7-UP) of the spray dried concentratedsupernatant and modified concentrated supernatant, produced by theprocedures of Example 1, was determined using a modification of theprocedure of Morr et al, J. Food Sci. 50:1715-1718, as described abovein Example 7, but with the soft drink replacing the water, a proteinconcentration of 2 w/v and no pH adjustment performed.

The results obtained are set forth in the following Table XIV:

TABLE XIV Batch Product Solubility (%) SA034-J12-04A Concentratedsupernatant 92.3 SA034-J12-04A Modified concentrated supernatant 94.9SA035-J14-04A Concentrated supernatant 96.1 SA035-J14-04A Modifiedconcentrated supernatant 94.4

As may be seen from the results of Table XIV, the dried isolate frommodified concentrated supernatant (C200H) and concentrated supernatant(C200) had similar solubilities in the soft drink.

However, as may be seen from the results of Example 11 below, theclarity of the solution prepared from the dried isolate from modifiedconcentrated supernatant was far superior.

Example 10

This Example contains an evaluation of the solubility of the canolaprotein isolates produced in Examples 4 and 6 in a soft drink and asports drink.

The solubility in a soft drink (Sprite) or a sports drink (Gatorade) ofthe spray dried concentrated supernatant (C200) and modified products(C200H and C200HS) produced by the procedures of Example 4 andisoelectrically precipitated supernatant (C200IS) produced by theprocedures of Example 6, were determined using a modification of theprocedure of Morr et al., J. Food Sci. 50:1715-1718, as described abovein Example 7 but with the soft drink or sport drink replacing the water,a protein concentration of 2% w/v and no pH adjustment performed.

The results obtained are set forth in the following Table XV:

TABLE XV Solubility Solubility (%) in (%) in carbonated non-carbonatedBatch Product soft drink sports drink SA033-E10-04A Concentrated 91.585.2 supernatant (C200) MS043-E04-05A Concentrated 94.2 84.8 supernatant(C200) MS043-E04-05A Modified concentrated 92.0 96.3 supernatant (C200H)SD061-K07-05A Modified concentrated 99.3 97.0 supernatant (C200H)SD062-L18-05A Modified concentrated 100 98.2 supernatant (C200H)SD061-I26-06A Supernatant heat treated 100 98.7 then concentrated(C200HS) SD062-K20-06A Supernatant partially 97.3 95.6 concentrated,then heat treated, then concentrated (C200HS) SA-077-K07-07AIsoelectrically 95.2 95.7 precipitated supernatant (C200IS)

As can be seen from the results of Table XV, the dried isolate frommodified concentrated supernatant (C200H), heat treated thenconcentrated supernatant (C200HS) and isoelectrically precipitatedsupernatant (C200IS) were notably more soluble in a soft drink and asports drink than the dried isolate from concentrated supernatant(C200).

Example 11

This Example contains an evaluation of the clarity in solutions of spraydried isolates produced in Example 1 dissolved in a soft drink.

The clarity in a soft drink (7-UP) of solutions of spray dried isolatesfrom modified concentrated supernatant and concentrated supernatant,produced as described in Example 1, was determined. Clarity was assessedby measuring the absorbance of visible light at 600 nm by a solution of2% w/v protein in a colourless, transparent, commercial carbonated softdrink. The lower the absorbance reading, the better light was beingtransmitted and the better the clarity of the solution.

The results obtained are set forth in the following Table XVI:

TABLE XVI Batch Product A600 SA034-J12-04A Concentrated supernatant0.544 SA034-J12-04A Modified concentrated supernatant 0.150SA035-J14-04A Concentrated supernatant 1.920 SA035-J14-04A Modifiedconcentrated supernatant 0.367

As may be seen from the results of Table XVI, the clarity of thesolution prepared from the spray dried isolate from modifiedconcentrated supernatant (C200H) was far superior to that prepared fromthe spray dried concentrated supernatant (C200).

Example 12

This Example contains an evaluation of the clarity in solutions of thespray dried concentrated supernatant (C200), modified products (C200Hand C200HS) produced by the procedures of Example 4 and isoelectricallyprecipitated supernatant (C200IS) produced by the procedures of Example6 dissolved in water at various pH values.

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with NaOH or HCl. A sample was also prepared at native pH.For the pH adjusted samples, the pH was measured and corrected two timesduring the 60 minutes stirring. After the 60 minutes of stirring, thesample was made up to 50 ml total volume with RO water, yielding a 1%w/v protein dispersion. The clarity of the solutions was determined bymeasuring the absorbance at 600 nm with pure water used to blank thespectrophotometer. The lower the absorbance reading, the greater theclarity.

The results obtained are set forth in the following Table XVII:

TABLE XVII A600 Native Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pHSA033-E10-04A Concentrated 2.117 2.167 2.221 2.244 2.267 2.364 2.278supernatant (C200) MS043-E04-05A Concentrated 1.748 1.902 1.937 2.0101.688 1.613 1.834 supernatant (C200) MS043-E04-05A Modified 0.185 0.2100.237 0.493 0.518 0.722 0.610 concentrated supernatant (C200H)SD061-K07-05A Modified 0.080 0.085 0.079 0.212 0.432 0.507 0.402concentrated supernatant (C200H) SD062-L18-05A Modified 0.047 0.0490.046 0.050 0.306 0.553 0.058 concentrated supernatant (C200H)SD061-I26-06A Supernatant heat 0.043 0.050 0.052 0.367 0.649 0.426 0.791treated then concentrated (C200HS) SD062-K20-06A Supernatant 0.044 0.0500.049 0.146 0.482 0.520 0.539 partially concentrated, then heat treated,then concentrated (C200HS) SA077-K07-07A Isoelectrically 0.040 0.0440.052 0.199 0.584 0.324 0.050 precipitated supernatant (C200IS)

As can be seen from the results of Table XVII, the dried isolate frommodified concentrated supernatant (C200H), heat treated thenconcentrated supernatant (C200HS) and isoelectrically precipitatedsupernatant (C200IS) produced solutions with notably greater clarity inwater at various pH values than the dried isolate from concentratedsupernatant (C200). The difference was most pronounced at the low pHvalues.

Example 13

This Example contains an evaluation of the clarity in solutions of spraydried isolates produced in Examples 4 and 6 dissolved in a soft drink ora sports drink.

The clarity in a soft drink (Sprite) or a sports drink (Gatorade) ofsolutions of spray dried isolates produced as described in Examples 4and 6, was determined. Clarity was assessed by measuring the absorbanceof visible light at 600 nm by a solution of 2% w/v protein in acolourless, transparent, commercial carbonated soft drink (Sprite) or acoloured, commercial non-carbonated sports drink (Gatorade). Thespectrophotometer was blanked with a pure sample of the appropriatedrink prior to measurement of the clarity of a protein containingsample. The lower the absorbance reading, the better light was beingtransmitted and the better the clarity of the solution.

The results obtained are set forth in the following Table XVIII:

TABLE XVIII A600 in A600 in carbonated non-carbonated Batch Product softdrink sports drink SA033-E10-04A Concentrated 2.716 2.808 supernatant(C200) MS043-E04-05A Concentrated 2.248 2.514 supernatant (C200)MS043-E04-05A Modified concentrated 0.397 0.954 supernatant (C200H)SD061-K07-05A Modified concentrated 0.099 0.363 supernatant (C200H)SD062-L18-05A Modified concentrated 0.079 0.344 supernatant (C200H)SD061-I26-06A Supernatant heat treated 0.081 0.326 then concentrated(C200HS) SD062-K20-06A Supernatant partially 0.092 0.311 concentrated,then heat treated, then concentrated (C200HS) SA077-K07-07AIsoelectrically 0.040 0.168 precipitated supernatant (C200IS)

As may be seen from the results of Table XVIII, the clarity of thesolutions prepared from the spray dried isolate from modifiedconcentrated supernatant (C200H), heat treated then concentratedsupernatant (C200HS) and isoelectrically precipitated supernatant(C200IS) were far superior to those prepared from the spray driedconcentrated supernatant (C200).

Example 14

This Example contains the pH values of common beverages.

The pH value of common beverages was determined by a pH meter and theresults obtained are set forth in the following Table XIX:

TABLE XIX pH values of common beverages Beverage pH Coca Cola Classic2.68 Diet Coke 3.41 7-Up 3.60 Sprite 3.24 Mountain Dew 3.10 Canada DryGinger Ale 2.85 Iced Tea (tea flavoured drink) 3.19 Snapple Peach IcedTea (contains natural tea) 2.96 Club Soda 4.61 Orange Gatorade 2.98Fruit Punch Gatorade 3.05 Lemon Lime Gatorade 2.97 Green Tea SoBe 2.72apple juice (not from concentrate) 3.62 orange juice (from concentrate)3.91 grape juice (from concentrate) 3.20 tropical juice blend (apple,orange, pineapple, 3.77 passionfruit, mango) (from concentrate) apple,orange, passionfruit juice blend (from 3.69 concentrate) fruit punch2.93 cranberry cocktail 2.77

Example 15

This Example contains a further evaluation of the solubility of thespray dried concentrated supernatant (C200), modified products (C200Hand C200HS) and isoelectrically-precipitated supernatant (C200IS)produced by procedures of Examples 1, 4 and 6.

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with NaOH or HCl. A sample was also prepared at native pH.For the pH adjusted samples, the pH was measured and corrected two timesduring the 60 minutes stirring. After the 60 minutes of stirring, thesamples were made up to 50 ml total volume with RO water, yielding a 1%w/v protein dispersion. Samples (20 ml) of the dispersions were thentransferred to pre-weighed centrifuge tubes that had been dried in a105° C. oven for 30 minutes then cooled in a desiccator and the tubescapped. The samples were centrifuged at 7800 g for 10 minutes, whichsedimented insoluble material and yielded a clear supernatant. Thesupernatant and the tube lids were discarded and the pellet materialdried overnight in an oven set at 55° C. The next morning the tubes weretransferred to a desiccator and allowed to cool. The weight of drypellet material was recorded. The dry weight of the initial proteinpowder was calculated by multiplying the weight of powder used by afactor of ((100−moisture content of the powder (%))/100). Solubility ofthe product was then calculated as:

Solubility (%) (1−(weight dry insoluble pellet material/(2/5×initialweight dry protein powder)))×100

The results obtained are set forth in the following Table XX:

TABLE XX Solubility (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat.pH SA034- Concentrated 94.6 96.1 95.8 95.0 84.2 88.7 88.7 J12-04Asupernatant (C200) SA034- Modified 99.0 97.7 96.0 97.2 87.5 89.5 89.9J12-04A concentrated supernatant (C200H) SA035- Concentrated 96.2 95.796.0 93.3 86.2 87.5 86.7 J14-04A supernatant (C200) SA035- Modified 97.197.3 96.6 97.2 87.9 87.4 90.8 J14-04A concentrated supernatant (C200H)SA033- Concentrated 91.4 90.0 91.8 91.9 81.1 84.9 89.3 E10-04Asupernatant (C200) MS043- Concentrated 93.9 92.9 93.5 91.9 86.1 82.787.5 E04-05A supernatant (C200) SD061- Modified 98.8 97.6 97.4 95.3 91.093.3 94.8 K07-05A concentrated supernatant (C200H) SD061- Supernatantheat 97.5 97.6 98.4 95.8 88.2 92.7 88.1 I26-06A treated thenconcentrated (C200HS) SD062- Supernatant partially 98.3 98.2 97.9 97.691.9 92.2 93.4 K20-06A concentrated, then heat treated, thenconcentrated (C200HS) SA07- Isoelectrically 99.4 100 99.3 97.8 96.3 98.599.9 K07-07A precipitated supernatant (C200IS)

As can be seen from the results of Table XX, the dried isolate frommodified concentrated supernatant (C200H), heat treated thenconcentrated supernatant (C200HS) and isoelectrically precipitatedsupernatant (C200IS) were notably more soluble in water at various pHvalues than the dried isolate from concentrated supernatant (C200).

Example 16

This Example contains an evaluation of the heat stability at pH 7 of thespray dried concentrated supernatant (C200), modified product (C200H)and isoelectrically-precipitated supernatant (C200IS) produced byprocedures of Examples 1, 4 and 6.

Sufficient protein powder to supply 1.6 g of protein was weighed into abeaker and then approximately 15 g of reverse osmosis (RO) purifiedwater was added and the mixture stirred for a total of 60 minutes usinga magnetic stirrer. As necessary, the product was also manuallydispersed with a stirring rod as magnetic stirring proceeded. After 30minutes of stirring the pH of the sample was determined and adjusted to7 using NaOH or HCl as necessary. The sample was then stirred for anadditional 30 minutes. After the 60 minutes of stirring, the pH waschecked and readjusted to 7 if necessary then the sample was made up to20 g total weight with RO water, yielding an 8% w/w protein dispersion.The dispersions were transferred to pre-weighed centrifuge tubes thathad been dried in a 105° C. oven for 30 minutes then cooled in adesiccator. The weight of dispersion in the tubes was recorded and thenthe samples were capped and heat treated by placing them in a 90° C.water bath for 30 minutes. The water level was maintained higher thanthe sample liquid level throughout the heating process. After the heattreatment the samples were immediately cooled by placing them in an icewater bath. The samples were then placed in the refrigerator overnight.The next morning the samples were centrifuged three times each at 7800 gfor 10 minutes. The supernatants were decanted from the tubes (note someminor losses of pellet material were observed for certain samples wherecompletely compact pellets were not obtained) and the tube lidsdiscarded. The pellet material was dried by placing the open tubes in a55° C. oven overnight. The next morning the samples were transferred tothe desiccator and allowed to cool. The weight of dried pellet materialwas measured. The dry weight of the initial protein powder wascalculated by multiplying the weight of powder used by a factor of((100−moisture content of the powder (%))/100). The effect of heattreatment on product solubility was then calculated as:

% of powder insoluble after heat treatment=(weight dry insoluble pelletmaterial/weight dispersion in centrifuge tube/20)×initial weight dryprotein powder))×100

The results obtained are set forth in the following Table XXI:

TABLE XXI % of initial powder insoluble Sample after heat treatmentSA034-J12-04A C200 22.8 SA034-J12-04A C200H 12.3 SA035-J14-04A C200 18.6SA035-J14-04A C200H 12.7 SA033-E10-04A C200 42.2 SD061-K07-05A C200H14.6 SA077-K07-07A C200IS 5.8

As may be seen from the results of Table XXI, the heat stabilities ofthe solutions prepared from the spray dried isolates from modifiedconcentrated supernatant (C200H) and from isoelectrically precipitatedsupernatant (C200IS) were superior to those prepared from the spraydried concentrated supernatant (C200).

Example 17

This Example contains an evaluation of the heat stability at pH 6 of thespray dried concentrated supernatant (C200), modified products (C200Hand C200HS) and isoelectrically precipitated supernatant (C200IS)produced by procedures of Examples 1, 4 and 6.

Sufficient protein powder to supply 0.3 g of protein was weighed into abeaker and then approximately 25 g of reverse osmosis (RO) purifiedwater was added and the mixture stirred for a total of 60 minutes usinga magnetic stirrer. After 30 minutes of stirring the pH of the samplewas determined and adjusted to 6 using NaOH or HCl as necessary. Thesample was then stirred for an additional 30 minutes. After the 60minutes of stirring, the pH was checked and readjusted to 6 if necessarythen the sample was made up to 30 g total weight with RO water, yieldinga 1% w/w protein dispersion. The dispersions were transferred topre-weighed centrifuge tubes that had been dried in a 105° C. oven for30 minutes then cooled in a desiccator. The weight of dispersion in thetubes was recorded and then the samples were capped and heat treated byplacing them in a 85° C. water bath for 10 minutes. The water level wasmaintained higher than the sample liquid level throughout the heatingprocess. After the heat treatment, the samples were immediately cooledby placing them in an ice water bath. The samples were then centrifugedat 7800 g for 10 minutes. The supernatants were decanted from the tubesand the tube lids discarded. The pellet material was dried by placingthe open tubes in a 55° C. oven overnight. The next morning the sampleswere transferred to the desiccator and allowed to cool. The weight ofdry pellet material was measured. The dry weight of the initial proteinpowder was calculated by multiplying the weight of powder used by afactor of ((100−moisture content of the powder (%))/100). The effect ofheat treatment on product solubility was then calculated as:

% of powder insoluble after heat treatment=(weight dry insoluble pelletmaterial/((weight dispersion in centrifuge tube/30)×initial weight dryprotein powder))×100

The results obtained are set forth in the following Table XXII:

TABLE XXII % of initial powder insoluble Sample after heat treatmentSA034-J12-04A C200 19.6 SA034-J12-04A C200H 9.0 SA035-J14-04A C200 27.5SA035-J14-04A C200H 13.7 SA033-E10-04A C200 30.4 SD061-I26-06A C200HS8.9 SA077-K07-07A C200IS 4.5

As can be seen from the results of Table XXII, the heat stabilities ofthe solutions prepared from the spray dried isolate from modifiedconcentrated supernatant (C200H), heat treated then concentratedsupernatant (C200HS) and isoelectrically precipitated supernatant(C200IS) were superior to those prepared from the spray driedconcentrated supernatant (C200).

Example 18

This Example contains an evaluation of the solubility at pH 3.5 and asalinity of 0.10M NaCl of spray dried concentrated supernatant (C200),modified product (C200H) and isoelectrically precipitated supernatant(C200IS) produced by the procedures of Examples 1, 4 and 6.

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then approximately 45 g of 0.1M NaCl was added. The contentsof the beaker were stirred for 30 minutes using a magnetic stirrer. ThepH was then adjusted to 3.5 by the addition of HCl. The sample was thenstirred for an additional 30 minutes. At this time the pH was checkedand readjusted to 3.5 if necessary then the sample weight was made up to50 g with additional 0.1M NaCl, yielding a 1% w/w protein dispersion.Samples (approximately 30 g) of the dispersions were then transferred topre-weighed centrifuge tubes that had been dried in a 105° C. oven for30 minutes then cooled in a desiccator. The weight of dispersion in thetubes was recorded and the tubes capped. The samples were centrifuged at7800 g for 10 minutes, which sedimented insoluble material. Thesupernatants were decanted from the tubes (note some very minor lossesof pellet material were observed for certain samples where completelycompact pellets were not obtained) and the tube lids discarded. Thepellet material was dried overnight in an oven set at 55° C. The nextmorning the tubes were transferred to a desiccator and allowed to cool.The weight of dry pellet material was recorded. The dry weight of theinitial protein powder was calculated by multiplying the weight ofpowder used by a factor of ((100−moisture content of the powder(%))/100). The effect of the pH and salt concentration on productsolubility was then calculated as:

% powder insoluble under test conditions=(weight dry insoluble pelletmaterial/((weight of initial sample in centrifuge tube/50)×initialweight dry protein powder))×100

The results obtained are set forth in the following Table XXIII:

TABLE XXIII % of initial powder insoluble Sample in 0.1M NaCl at pH 3.5SA034-J12-04A C200 4.1 SA034-J12-04A C200H 1.4 SA035-J14-04A C200 9.4SA035-J14-04A C200H 2.6 SA033-E10-04A C200 19.1 SD061-K07-05A-C200H 1.6SA077-K07-07A C200IS 0.8

As can be seen from the results of Table XXIII, the dried isolates frommodified concentrated supernatant (C200H) and isoelectricallyprecipitated supernatant (C200IS) were notably more soluble in saline atpH 3.5 than the dried isolate from concentrated supernatant (C200).

Example 19

This Example contains an evaluation of the surface hydrophobicity ofspray dried concentrated supernatant (C200), modified products (C200Hand C200HS) and isoelectrically-precipitated supernatant (C200IS)produced by the procedures of Examples 1, 4 and 6.

The surface hydrophobicities of the different isolates were determinedat room temperature using the surface hydrophobicity (S₀) test involvingthe hydrophobic dye: 1-aniline 8-naphthalene sulfonate (ANS). Therelative fluorescence intensity was measured using a spectrofluorimeterwith the hydrophobicity (S₀) for the proteins determined by calculationof the net relative fluorescence intensity (RFI) slope. The higher theS₀, the more hydrophobic the protein is, since the dye is attracted tohydrophobic sites on the protein molecule.

Since concentrated supernatant derived protein (C200) contains a higherpercentage of globulin (7S), this isolate was found to contain greaterhydrophobicity according to the ANS test. The canola globulins areconsidered to be quite hydrophobic and this was shown to be the case.The modified products produced lower values as expected with the reducedlevels of globulin proteins. The main component of both isolates is thealbumin napin. This protein is very polar resulting in a very lowsurface hydrophobicity.

The results are set forth in the following Table XXIV. The data isadjusted to reflect 100% (N×6.25) protein for each isolate.

TABLE XXIV Sample % 7S S_(0 100% N×6.25) SA033-E10-04A C200 32.18% 78.1SA034-J12-04A C200 13.13% 32.6 SA035-J14-04A C200 24.52% 58.1SA034-J12-04A C200H 3.55% 12 SA035-J14-04A C200H 7.55% 14.9SD061-I26-06A C200HS 4.05% 12.1 SA077-K07-07A C200IS 3.12% 17.6

As can be seen from the results of Table XXIV, the dried isolates frommodified concentrated supernatant (C200H), heat treated thenconcentrated supernatant (C200HS) and isoelectrically-precipitatedsupernatant (C200IS) were notably less hydrophobic than the driedisolate from concentrated supernatant (C200). Furthermore, the resultsshow that the surface hydrophobicity is linearly related to the contentof globulins.

Example 20

This Example contains an evaluation of the absorbance at 280 nm of 1mg/ml protein solutions of spray dried concentrated supernatant (C200),modified products (C200H and C200HS) and isoelectrically precipitatedsupernatant (C200IS) produced by procedures of Examples 1, 4 and 6.

Sufficient protein powder to supply 50 mg of protein was weighed into abeaker and then approximately 45 ml of 10 mM phosphate buffer, pH 6.66was added and the mixture stirred for a total of 60 minutes using amagnetic stirrer. After the 60 minutes of stirring the sample was madeup to 50 ml total volume with buffer, yielding a protein concentrationof 1 mg/ml. The samples were clarified by centrifugation at 7800 g for10 minutes then the supernatants were transferred to a quartz cuvetteand the absorbance at 280 nm read using the phosphate buffer as a blank.

The results obtained are set forth in the following Table XXV:

TABLE XXV Sample Absorbance at 280 nm SA034-J12-04A C200 0.694SA034-J12-04A C200H 0.656 SA035-J14-04A C200 0.710 SA035-J14-04A C200H0.654 SA033-E10-04A C200 0.700 SD061-I26-06A C200HS 0.668 SA077-K07-07AC200IS 0.611

As may be seen from the results of Table XXV, the solutions preparedfrom the spray dried concentrated supernatant (C200) absorbed morestrongly at 280 nm than the solutions prepared from modifiedconcentrated supernatant (C200H), heat treated then concentratedsupernatant (C200HS) and isoelectrically precipitated supernatant(C200IS).

Example 21

This Example illustrates amino acid analysis of the novel canola proteinisolate of the present invention (C200H) and the supernatant-derivedcanola protein isolate (C200).

Typical samples of C200H and C200 were analyzed for amino acid contentand the results are presented in the following Table XXVI:

TABLE XXVI Amino Acid composition of C200 and C200H grams/100 gramsAmino Acid Difference Amino Acid C200 C200H C200H vs C200 Aspartic 5.593.66 −1.93 Glycine 4.71 4.77 0.06 Valine 5.29 4.92 −0.37 Isoleucine 3.853.15 −0.70 Leucine 7.13 6.33 −0.80 Tryptophan 1.42 1.10 −0.32 Tyrosine2.50 1.84 −0.66 Phenylanine 3.57 2.70 −0.87 Arginine 7.25 6.66 −0.59Threonine 3.43 3.85 0.42 Serine 4.24 4.17 −0.07 Alanine 4.24 4.63 0.39Glutamic 24.07 26.08 2.01 Cysteine 3.09 4.01 0.92 Methionine 2.40 2.750.35 Histidine 3.19 3.13 −0.06 Lysine 5.26 6.98 1.72 Proline 8.78 9.270.49 Sum: 100.00 100.00

As may be may be seen from the results of Table XXVI, the C200H andsamples differed in amino acid content, there being an increased contentof glycine, threonine, alanine, cysteine, proline, lysine, methionineand glutamine-glutamate in the C200H sample. In addition, there is adecreased content of aspargine-aspartate, valine, leucine, tryptophan,tyrosine, phenylalanine, serine, isoleucine, arginine and histidine inthe C200H sample.

Example 22

This Example illustrates an alternative process of forming the novelcanola protein isolate of the invention (FIG. 1).

15 kg of canola oil seed meal was added to 100 L (15% w/v) of 0.15Msodium chloride solution containing 0.05 wt % ascorbic acid at ambienttemperature in a 350 L extraction tank and the mixture was agitated for30 minutes to provide a canola protein solution having a concentrationof 20 g/L. Bulk residual meal was removed by using a basket centrifugewith a 400 mesh bag and the separated bulk meal was discharged to waste.The canola protein solution was given a second pass through the basketcentrifuge using a 600 mesh bag to remove suspended fine particles. Theresulting canola protein solution was polished using a filter press with2 μm filter pads.

The clarified canola protein solution was subjected to anultrafiltration step using a spiral wound polyvinylidiene difluoride(PVDF) membrane with a molecular weight cut-off of 100,000 daltons atambient temperature to concentrate the canola protein solutioncontaining 7S and 12S proteins to a volume of 4.3 L and a proteinconcentration of 188 g/L. The permeate from the ultrafiltration stepcontained the 2S protein along with other low molecular weight species.

The concentrated canola protein solution (retentate) then was subjectedto a diafiltration step using the same membrane as for theultrafiltration using an aqueous 0.15M sodium chloride solutioncontaining 0.05 wt % ascorbic acid. The diafiltration medium was addedto the retentate at the same flow rate as permeate was removed from themembrane. The diafiltration was carried out with 5 retentate volumes ofdiafiltration medium.

A 1.25 L aliquot of the retentate from the ultrafiltration anddiafiltration operations was spray dried to provide a canola proteinisolate consisting predominantly of 7S protein, having a protein contentof 99.1 wt % (N×6.25, percent nitrogen values were determined using aLeco FP528 Nitrogen Determinator) d.b. and containing 18.21 wt % 2Sprotein, 74.55 wt % 7S protein and 7.24 wt % 12S protein.

The permeate from the ultrafiltration and diafiltration operations wassubjected to an ultrafiltration step using a spiral woundpolyethersulfone (PES) membrane with a molecular weight cut-off of 5000daltons to permit retention of 2S protein and to permit low molecularweight contaminants to pass through the membrane to waste. Thisultrafiltration step was effected at ambient temperature to concentratethe 2S-containing permeate from the first ultrafiltration step to 3 Lhaving a protein concentration of 125 g/L.

The concentrated canola 2S protein solution (retentate) then wassubjected to a diafiltration step using the same membrane as for theultrafiltration and using filtered tap water as the diafiltrationmedium. The water was added to the retentate at the same flow rate aspermeate was removed from the membrane. The diafiltration was carriedout with 5 retentate volumes of diafiltration medium.

The retentate from the diafiltration step was spray dried to provide acanola protein isolate consisting predominantly of 2S protein, having aprotein content of 105.8 wt % (N×6.25) d.b. and containing 96.7 wt % 2Sprotein, 3.3 wt % 7S protein and 0.04 wt % of 12S protein.

Example 23

This Example is a repeat of the process of Example 22, but on a largerscale.

150 kg of canola oil seed meal was added to 1000 L (15% w/v) of 0.15 Msodium chloride solution containing 0.05 wt % of ascorbic acid atambient temperature in a 10,000 L extraction tank and the mixture wasagitated for 30 minutes to provide a canola protein solution having aconcentration of 20.7 g/L. Bulk residual meal was removed by using avacuum filter belt and the separated meal was discharged to waste. Thecanola protein solution was clarified by using a disc centrifuge and thedesludged solids discharged to waste. The resulting canola proteinsolution was polished using a filter press with 2 μm filter padsfollowed by another one with 0.2 μm pads.

The clarified canola protein solution was subjected to ultrafiltrationusing two spiral wound PVDF membranes with a molecular weight cut-off of100,000 daltons to concentrate the canola protein solution containing 7Sand 12S proteins to 41.1 L having a protein concentration of 221 g/L.The permeate from the ultrafiltration step contained the 2S proteinalong with other low molecular weight species.

A 3 L aliquot of the retentate from the ultrafiltration operation wasspray dried to provide a canola protein isolate consisting predominantlyof 7S protein, having a protein content of 95.1 wt % (N×6.25) d.b. andcontaining 26.86 wt % of 2S protein, 66.22 wt % of 7S protein and 6.92wt % of 12S protein.

The permeate from the ultrafiltration operation was subjected to anultrafiltration step using two spiral wound PVDF membranes with amolecular weight cut-off of 5,000 daltons to permit retention of 2Sprotein and to permit low molecular weight contaminants to pass throughthe membrane to waste. This ultrafiltration step was effected at ambienttemperature to concentrate the 2S-containing permeate from the firstultrafiltration step to 25 L having a protein concentration of 24.2 g/L.

The retentate from the ultrafiltration step was spray dried to form anon-diafiltered canola protein product having a protein concentration of47.94 wt % (N×6.25) d.b. and containing 94.64 wt % of 2S protein, 5.36wt % of 7S protein and 0 wt % of 12S protein.

The low protein content of the latter canola protein product was due tothe absence of a diafiltration step to remove the salt and otherimpurities. Later bench diafiltration with this product gave resultsthat indicated the production of a canola protein isolate afterdiafiltration.

Example 24

This Example provides a comparison of the canola protein isolateproducts prepared according to the procedure of Example 23 with canolaprotein isolate products prepared by the micelle route.

A 34 L aliquot of the retentate from the first ultrafiltration stepdescribed in Example 23 was warmed to 29.8° C. and poured into chilledwater having a temperature of 3.7° C. at a ratio of 10 volumes of waterper volume of retentate. A white cloud of protein micelles immediatelyformed. The micelles were allowed to coalesce and settle overnight. Theaccumulated protein micellar mass was separated from supernatant and wasspray dried to provide a canola protein isolate having a protein contentof 107.4 wt % (N×6.25) d.b. and containing 3.80 wt % 2S protein, 85.88wt % 7S protein and 10.32 wt % 12S protein.

The supernatant from the PMM-settling step (365 L) was subjected to anultrafiltration step using a spiral wound PVDF membrane with a molecularweight cut-off of 5000 daltons to permit retention of 2S protein and 7Sprotein and to permit low molecular weight contaminants to pass throughthe membrane to waste. This ultrafiltration step was effected at ambienttemperature to concentrate the supernatant to 22 L having a proteincontent of 89.3 g/L.

The concentrated supernatant was spray dried to provide a canola proteinisolate consisting predominantly of 2S protein, having a protein contentof 95.51 wt % (N×6.25) d.b. and containing 84.01 wt % 2S protein, 15.51wt % 7S protein and 0.48 wt % of 12S protein.

SUMMARY OF DISCLOSURE

In summary of this disclosure, a novel canola protein isolate having anincreased content of 2S protein and a reduced quantity of 7S protein isprovided having utility in producing clear aqueous solutions,particularly in beverages. Modifications are possible within the scopeof the invention.

1. A composition comprising: a first isolated or purified protein component of a plant origin and a second isolated or purified protein component of a plant origin; wherein, upon heat treatment of an about 1% w/v dispersion of the composition in water at a pH of between about 6 to about 7 at about 85° C. for about 10 minutes, less than about 15% of the composition is insoluble.
 2. The composition of claim 1 wherein, upon said heat treatment, less than 10% of the composition is insoluble.
 3. The composition of claim 1 wherein, upon said heat treatment, substantially none of the composition is insoluble.
 4. The composition of claim 1 wherein the plant is canola, the first protein component comprises a 2S canola protein and the second protein component comprises a 7S canola protein.
 5. The composition of claim 4 wherein the 2S canola protein comprises at least about 85% by weight of the composition.
 6. The composition of claim 5 wherein the 2S canola protein component comprises at least about 95% by weight of the composition.
 7. The composition of claim 5 wherein the 7S canola protein comprises at least about 2% by weight of the composition.
 8. The composition of claim 1 wherein the composition has a protein content of at least about 90% by weight (N×6.25).
 9. The composition of claim 1 wherein the first protein component and the second component have not been hydrolyzed.
 10. A beverage comprising the composition of claim
 1. 11. The beverage of claim 10 having a pH of about 2.5 to about
 5. 12. The beverage of claim 11 wherein 12 fluid ounces of the beverage contain at least about 5 grams of the composition.
 13. An aqueous solution of the composition of claim
 1. 14. The aqueous solution of claim 13 having a clarity of an about 1 wt % aqueous solution over a pH range of about 2 to about 7 of less than about 1 when determined by measuring absorbance of visible light at 600 nm.
 15. A beverage comprising an isolated and purified protein of vegetable origin, wherein, upon heat treatment of an about 1% w/v dispersion of the protein in water at a pH of about 6 to about 7 at about 85° C. for about 10 minutes, less than about 15% of the protein is insoluble.
 16. The beverage of claim 15 wherein, upon said heat treatment, substantially none of the protein is insoluble.
 17. The beverage of claim 16, wherein the isolated and purified protein provides at least about 5 grams of protein per serving.
 18. A beverage comprising an isolated and purified protein of vegetable origin, wherein an about 1% w/v aqueous solution of said protein has a clarity on a pH range of about 2 to about 7 of less than about 1 when determined by measuring absorbance of visible light at 600 nm.
 19. The beverage of claim 18 wherein the isolated and purified protein provides at least about 5 grams of protein per serving.
 20. A composition having a protein content of at least about 90 wt % (N×6.25) on a dry weight basis (d.b.) and comprising: a first canola protein isolate component which is the 2S protein of canola, and a second canola protein isolate component which is the 7S protein of canola, said composition being formed by processing the aqueous supernatant from canola protein micelle formation and precipitation and having a solubility in aqueous media greater than a canola protein isolate derived directly from the aqueous supernatant from said canola protein micelle formation and precipitation, wherein said solubility is determined as described in Example 15 and/or Example
 18. 21. The composition of claim 20 wherein said composition has a surface hydrophobicity of less than about 25, determined as described in Example
 19. 22. The composition of claim 20 wherein said composition has an amino acid profile substantially as set forth in the column of Table XXVI headed C200H.
 23. The composition of claim 20 which has an absorbance at 280 nm, determined as described in Example 20, which is less than the canola protein isolate derived directly from the aqueous supernatant.
 24. The composition of claim 20 wherein, upon heat treatment of an about 1% w/v dispersion of the composition in water at a pH of between about 6 and about 7 at about 85° C. for about 10 minutes, less than 15% of the composition is insoluble.
 25. The composition of claim 24 wherein, upon said heat treatment, substantially none of the composition is insoluble.
 26. The composition of claim 20 wherein said 2S protein of canola comprises at least about 85% by weight of the composition.
 27. The composition of claim 26 wherein said 2S protein of canola comprises at least about 95 wt % of the composition.
 28. The composition of claim 26 wherein said 7S protein of canola comprises at least about 2% by weight of the composition.
 29. An aqueous solution of the composition of claim
 20. 30. The aqueous solution of claim 29 having a clarity of an about 1 wt % aqueous solution over a pH range of about 2 to about 7 of less than about 1 when determined by measuring absorbance of visible light at 600 nm.
 31. A beverage comprising the composition of claim
 20. 32. The beverage of claim 31 having a pH of about 2.5 to about
 5. 33. The beverage of claim 32 wherein 12 fluid ounces of the beverage comprises at least about 5 grams of the composition.
 34. A canola protein isolate consisting predominantly of 2S canola protein having a protein content of at least about 90 wt % (N×6.25) on a dry weight basis (d.b.) and having an increased proportion of 2S canola protein and a decreased proportion of 7S canola protein when compared to canola protein isolates consisting predominantly of 2S canola protein and derived from aqueous supernatant from canola protein micelle formation and precipitation.
 35. The canola protein isolate of claim 34 which is derived by heat treatment of said aqueous supernatant.
 36. The canola protein isolate of claim 34 which is derived from a selective membrane procedure in which an aqueous canola protein solution derived from canola oil seed meal and containing 12S, 7S and 2S canola proteins is subjected to a first selective membrane technique which selectively retains 12S and 7S canola proteins in a retentate and permits 2S protein to pass through the membrane as a permeate, the permeate is subjected to a second selective membrane technique which selectively retains 2S canola protein and permits low molecular weight contaminants to pass through the membrane as a permeate, and the retentate from the second selective membrane technique is dried.
 37. The canola protein isolate of claim 34 having a protein content of at least about 100 wt % (N×6.25) d.b.
 38. A canola protein isolate having a protein content of at least about 90 wt % (N×6.25) on a dry weight basis (d.b.) and containing at least about 85 wt % of 2S canola protein and less than about 15 wt % of 7S canola protein of the canola proteins present in the isolate.
 39. The canola protein isolate of claim 38 wherein the isolate contains at least about 90 wt % of 2S canola protein and less than about 10 wt % of 7S canola protein of the canola proteins present in the isolate.
 40. The canola protein isolate of claim 38 having a protein content of at least about 100 wt % (N×6.25) d.b.
 41. A process for the preparation of a canola protein isolate having an increased proportion of 2S canola protein, which comprises: (a) providing an aqueous solution of 2S and 7S proteins consisting predominantly of 2S protein, (b) heat treating the aqueous solution to cause precipitation of 7S canola protein, (c) removing precipitated 7S protein from the aqueous solution, and (d) recovering a canola protein isolate having a protein content of at least about 90 wt % (N×6.25) d.b. and having an increased proportion of 2S canola protein.
 42. The process of claim 41 wherein said heat treatment step is effected under temperature and time conditions sufficient to precipitate at least about 50 wt % of the 7S canola protein present in said aqueous solution.
 43. The process of claim 42 wherein said heat treatment step precipitates the 7S canola protein by at least about 75% of the 7S canola protein present in said aqueous solution.
 44. The process of claim 41 wherein said heat treatment step is effected by heating the aqueous solution for about 1 second to about 30 minutes at a temperature of about 70° to about 120° C.
 45. The process of claim 44 wherein said heat treatment step is effected for about 5 to about 15 minutes at a temperature of about 75° to about 105° C.
 46. The process of claim 41 wherein said aqueous solution of 2S and 7S canola proteins is supernatant, partially concentrated supernatant or concentrated supernatant from canola protein micelle formation and precipitation.
 47. The process of claim 46 wherein said canola protein micelle formation is effected by: (a) extracting canola oil seed meal at a temperature of at least about 5° C. to cause solubilization of protein in said canola oil seed meal and to form an aqueous protein solution, (b) separating said aqueous protein solution from residual oil seed meal, (c) increasing the concentration of said aqueous protein solution to at least about 200 g/L while maintaining the ionic strength substantially constant by a selective membrane technique to provide a concentrated protein solution, (d) diluting said concentrated protein solution into chilled water having a temperature of below about 15° C. to cause the formation of the protein micelles, and (e) separating supernatant from settled protein micellar mass.
 48. The process of claim 47 wherein said supernatant is concentrated to a protein concentration of about 100 to about 300 g/L prior to said heat treatment.
 49. The process of claim 48 wherein said supernatant is concentrated to a protein concentration of about 200 to about 300 g/L.
 50. The process of claim 48 wherein said concentration step is effected by ultrafiltration using a membrane having a molecular weight cut-off of about 3,000 to about 100,000 daltons.
 51. The process of claim 50 wherein the concentrated supernatant resulting from ultrafiltration is subjected to diafiltration prior to said heat treatment step.
 52. The process of claim 51 wherein said diafiltration step is effected using from about 2 to about 20 volumes, preferably about 5 to about 10 volumes, of water, saline or acidified water using a membrane having a molecular weight cut-off of about 3,000 to about 100,000 daltons.
 53. The process of claim 41 wherein said heat treated solution post said removal of 7S protein, is acidified prior to drying.
 54. The process of claim 41 wherein said canola protein isolate has a protein content of at least about 100 wt % (N×6.25) d.b.
 55. The process of claim 41 further comprising: (e) formulating said canola protein isolate as an aqueous beverage composition.
 56. A process for the preparation of canola protein isolate, which comprises: (a) providing an aqueous canola protein solution derived from canola oil seed meal and containing 12S, 7S and 2S canola proteins, (b) increasing the protein concentration of the aqueous solution using a selective membrane technique which is effective to retain 7S and 12S canola proteins in a retentate and to permit 2S protein to pass through the membrane as a permeate to provide a concentrated protein solution, (c) drying the retentate from step (b) to provide a canola protein isolate consisting predominantly of 7S canola protein and having a protein content of at least about 90 wt % (N×6.25) on a dry weight basis (d.b.), (d) increasing the concentration of the permeate from step (b) using a selective membrane technique which is effective to retain 2S canola protein in a retentate and to permit low molecular weight contaminants to pass through the membrane in a permeate, and (e) drying the retentate from step (d) to provide a canola protein isolate consisting predominantly of 2S protein and having a protein content of at least about 90 wt % (N×6.25) d.b.
 57. The process of claim 56 wherein said aqueous canola protein solution is provided by extracting canola oil seed meal at a temperature of at least about 5° C. to cause solubilization of protein in said canola oil seed meal and to form an aqueous protein solution having a protein content of about 5 to about 40 g/L and a pH of about 5 to about 6.8 and separating the aqueous protein solution from the residual oil seed meal.
 58. The process of claim 56 wherein step (b) is effected by concentrating the aqueous solution to a protein content of at least about 200 g/L while maintaining the ionic strength substantially constant using an ultrafiltration membrane having a molecular weight cut-off of about 30,000 to about 1,000,000 daltons, preferably about 50,000 to about 100,000 daltons, to provide the concentrated protein solution.
 59. The process of claim 58 wherein the concentrated protein solution is subjected to a diafiltration step using about 2 to about 20, preferably about 5 to about 10, volumes of diafiltration solution.
 60. The process of claim 56 wherein step (d) is effected by increasing the concentration of the permeate to a protein concentration of about 100 to about 300 g/L, preferably about 200 to about 300 g/L, using a membrane having a molecular weight cut-off of about 3,000 to about 30,000 daltons, preferably about 5,000 to about 10,000 daltons, to provide the retentate.
 61. The process of claim 60 wherein the retentate is subjected to a diafiltration step using about 2 to about 20, preferably about 5 to about 10, volumes of diafiltration solution, using water, acidified water or dilute saline.
 62. The process of claim 56 wherein at least one of the canola protein isolates produced in steps (c) and (e) has a protein content of at least about 100 wt %.
 63. The process of claim 56 further comprising: (f) formulating said canola protein isolate as an aqueous beverage composition.
 64. An aqueous solution of the canola protein isolate of claim
 34. 65. The aqueous solution of claim 64 which is a canola protein isolate fortified beverage.
 66. An aqueous solution of the canola protein isolate of claim
 38. 67. The aqueous solution of claim 66 which is a canola protein isolate fortified beverage. 